Use of glucocorticoid receptor modulators to potentiate checkpoint inhibitors

ABSTRACT

This invention provides a method that combines a checkpoint inhibitor and a glucocorticoid receptor modulator to treat cancer, e.g., a checkpoint inhibitor sensitive cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase under 35 U.S.C. § 371 of PCTApplication No. PCT/US2017/019948, filed Feb. 28, 2017, which claims thebenefit of, and priority to, U.S. Provisional Patent Application Ser.No. 62/302,106 filed Mar. 1, 2016, and claims the benefit of, andpriority to, U.S. Provisional Patent Application Ser. No. 62/320,276filed Apr. 8, 2016. The content of each of the aforementionedapplications is herein incorporated by reference in its entirety.

BACKGROUND

Cancer is a group of varied diseases characterized by uncontrolledgrowth and spread of abnormal cells. The pathways regulating celldivision and or cellular communication become altered in cancer cellssuch that the effects of these regulatory mechanisms in controlling andlimiting cell growth fails or is bypassed. Through successive rounds ofmutation and natural selection, a group of abnormal cells, generallyoriginating from a single mutant cell, accumulates additional mutationsthat provide selective growth advantage over other cells, and thusevolves into a cell type that predominates in the cell mass. As thecancer cells further evolve, some become locally invasive and thenmetastasize to colonize tissues other than the cancer cell's tissue oforigin. This property along with the heterogeneity of the tumor cellpopulation makes cancer a particularly difficult disease to treat anderadicate.

Traditional cancer therapies take advantage of the higher proliferativecapacity of cancer cells and their increased sensitivity to DNA damage:Ionizing radiation, including γ-rays and x-rays, and cytotoxic agents,such as bleomycin, vinblastine, cyclophosphamide, 5′-fluorouracil, andmethotrexate rely upon a generalized damage to DNA and destabilizationof chromosomal structure which eventually leads to destruction of cancercells. These treatments are particularly effective for those types ofcancers that have defects in cell cycle checkpoint, which limits theability of these cells to repair damaged DNA before undergoing celldivision. The non-selective nature of these treatments, however, oftenresults in severe and debilitating side effects. The systemic use ofthese drugs may result in damage to normally healthy organs and tissues,and compromise the long-term health of the patient.

Recently, immunotherapy targeting immune checkpoint signaling pathwayshas been shown to be effective in treating cancer. These pathwayssuppress immune response and are crucial for maintaining self-tolerance,modulating the duration and amplitude of physiological immune responsesin peripheral tissues, and minimizing collateral tissue damage. It isbelieved that tumor cells can activate the immune checkpoint signalingpathways to decrease the effectiveness of the immune response againsttumor tissues. Many of these immune checkpoint signaling pathways areinitiated by interactions between checkpoint proteins present on thesurface of the cells participating in the immune responses, e.g., Tcells, and their ligands, thus they can be readily blocked by agents ormodulated by recombinant forms of the checkpoint proteins or ligands orreceptors. The agents blocking the immunosuppression pathway induced bycheckpoint proteins are commonly referred to as checkpoint inhibitorsand a few have been commercialized. Cytotoxic T-lymphocyte-associatedantigen 4 (CTLA4) antibodies, blocking the immunosuppression pathway bythe checkpoint protein CTLA4, were the first of this class ofimmunotherapeutics to achieve US Food and Drug Administration (FDA)approval. Clinical findings with blockers of additionalimmune-checkpoint proteins, such as programmed cell death protein 1(PD-1), indicate broad and diverse opportunities to enhance anti-tumorimmunity with the potential to produce durable clinical responses.

Glucocorticoid receptor (GR) mediated signaling pathways have dynamicbiologic effects involving different components of the immune system andtheir in vivo effects are unpredictable. For example, glucocorticoidshave been reported to have both immunosuppressive effects such as,suppression of proflammatory cytokines, promotion of anti-inflammatorycytokines, inhibition of dendritic cells, suppression of natural killercells, promotion of T-regulatory cells, and induction of T cellapoptosis,—and immune-enhancing effects, See Hinrichs J. Immunother.2005: 28 (6): 517-524. The effects of GR mediated signaling pathway oncancer cells is likewise elusive. On one hand, it is believed thatactivating the GR signaling pathways induce apoptosis in certain typesof cancer cells, for example, malignant lymphoid cancers. SeeSchlossmacher, J. Endocrino. (2011). On the other hand, it has also beenreported that agents blocking the GR signaling pathway can potentiatechemotherapy in killing cancer cells. See U.S. Pat. No. 9,149,485. Thiscurrent invention uses a novel combination therapy that targets both thecheckpoint signaling pathway and GR signaling pathway to treat patientssuffering from a tumor load.

SUMMARY

The cancer treatment method disclosed herein includes administering to apatient suffering from a tumor load a therapeutic amount of a checkpointinhibitor and a selective glucocorticoid receptor modulator (SGRM) in anamount effective to potentiate the activity of the checkpoint inhibitor.The combination therapy of the checkpoint inhibitor and SGRM providessuperior tumor load reduction compared to treatment with a checkpointinhibitor alone.

In some cases, the checkpoint inhibitor is an antibody against at leastone checkpoint protein, e.g., PD-1, CTLA-4, PD-L1 or PD-L2. In somecases, the checkpoint inhibitor is an antibody that is effective againsttwo or more of the checkpoint proteins selected from the group of PD-1,CTLA-4, PD-L1 and PD-L2.

In some cases, the checkpoint inhibitor is a small molecule, non-proteincompound that inhibits at least one checkpoint protein. In oneembodiment, the checkpoint inhibitor is a small molecule, non-proteincompound that inhibits a checkpoint protein selected from the groupconsisting of PD-1, CTLA-4, PD-L1 and PD-L2.

In one embodiment, the SGRM is mifepristone. In some cases, the SGRM isa compound having a non-steroidal backbone. In some cases, the SGRM is afused azadecalin.

In some cases, the SGRM is CORT 125134, i.e.,(R)-(1-(4-fluorophenyl)-6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone,which has the following structure:

In some cases, the SGRM is mifepristone.

In some cases, the SGRM is CORT125281, i.e.,((4aR,8aS)-1-(4-fluorophenyl)-6-((2-methyl-2H-1,2,3-triazol-4-yl)sulfonyl)-4,4a,5,6,7,8,8a,9-octahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone,which has the following structure:

In one embodiment, the cancer expresses the glucocorticoid receptor(GR⁺).

In some cases, the cancer is a GR⁺ cancer and the cancer is selectedfrom the group consisting of breast cancer, prostate cancer, melanoma,sarcoma, renal cell cancer, head and neck cancer, hepatocellular cancer,glioblastoma, cervical cancer, neuroendocrine cancer, bladder cancer,prostate cancer, esophageal cancer, mesothelioma, lung cancer, ovariancancer, pancreatic cancer, gall bladder cancer, gastric cancer,endometrial cancer, and colon cancer.

In one embodiment, the checkpoint inhibitor and SGRM areco-administered. In a preferred embodiment, the SGRM is CORT125134 andthe checkpoint inhibitor is an antibody against PD-1.

In some embodiments, provided herein is a SGRM for use in combinationwith a checkpoint inhibitor in a method of treating a patient hosting atumor load, the method comprising administering to a patient sufferingfrom a tumor load a therapeutic amount of a checkpoint inhibitor and aselective glucocorticoid receptor modulator (SGRM) in an amounteffective to potentiate the activity of the checkpoint inhibitor. Inaddition, all the related embodiments described above are also includedin these embodiments of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the tumor growth data from three (3) groups of mice thatwere treated with 1) anti-PD-1 vehicle (PBS, 10 ml/kg), i. p. twice aweek and the CORT125134 vehicle p.o. (10% DMSO, 0.1% Tween 80 and 89.9%HPMC (0.5%), 10 ml/kg) daily; 2) the mouse anti PD-1 antibody (cloneRPM1-14, 10 mg/kg) i. p. twice a week; and 3) the mouse anti PD-1antibody (clone RPM1-14, 10 mg/kg) i. p. twice a week and CORT125134 (aSGRM) (30 mg/kg) orally on a daily basis, respectively. The result showsthat the combination of anti PD-1 and CORT125134 is superior to both theanti PD-1 group and the vehicle group in reducing tumor growth.

FIG. 2 shows the mean tumor volume for each group of ten mice plottedagainst the number of days of tumor growth since initiation of thetreatment. Group I mice were dosed with the anti-PD-1 vehicle and theCORT125281 vehicle; Group II mice were dosed with mouse anti PD-1antibody; and Group III mice were dosed with mouse anti PD-1 antibodyand CORT125281. The combination of anti PD-1 and CORT125281 is superiorto both the anti PD-1 group and the vehicle group in reducing tumorgrowth.

FIG. 3 shows the mean tumor volume for each group of ten mice plottedagainst the number of days of tumor growth since initiation of thetreatment. Group I mice were dosed with the anti-CTLA4 vehicle and theCORT125134 vehicle; Group II mice were dosed with the mouse anti CTLA4antibody; Group III mice were dosed with mouse anti CTLA4 antibody andCORT125134; Group IV mice were dosed with mouse anti CTLA4 antibody andCORT125281. The combination of anti CTLA4 with CORT125134 and thecombination of anti CTLA4 with CORT125281 were each superior to both theanti CTLA4 group and the vehicle group in reducing tumor growth.

DETAILED DESCRIPTION

A. Introduction

This method disclosed herein can be used to treat a patient hosting atumor load by administering at least one SGRM and at least onecheckpoint inhibitor. The checkpoint inhibitor is administered in anamount that is effective to treat the cancer when administered alone orin combination with a SGRM. The SGRM is administered in an amount thatis effective to potentiate the checkpoint inhibitor's activity ofblocking the checkpoint signaling pathways. In some cases, the tumorload is caused by a checkpoint inhibitor sensitive cancer.

B. Definitions

As used herein, the term “subject” or “patient” refers to a human ornon-human organism. Thus, the methods and compositions described hereinare applicable to both human and veterinary disease. In certainembodiments, subjects are “patients,” i.e., living humans that arereceiving medical care for a disease or condition. This includes personswith no defined illness who are being investigated for signs ofpathology. Preferred are subjects who have an existing diagnosis of aparticular cancer which is being targeted by the compositions andmethods of the present invention. In some cases, a subject may sufferfrom one or more types of cancer simultaneously, at least one of whichis targeted by the compositions and methods of the present invention.Preferred cancers for treatment with the compositions described hereininclude, but are not limited to prostate cancer, renal carcinoma,melanoma, pancreatic cancer, cervical cancer, ovarian cancer, coloncancer, head & neck cancer, lung cancer, sarcoma, breast cancer,hepatocellular tumor, glioblastoma, neuroendocrine tumor, bladdercancer, gall bladder cancer, gastric cancer, endometrial cancer, andmesothelioma.

As used herein, the term “tumor load” or “tumor burden” generally refersto the number of cancer cells, the size of a tumor, or the amount ofcancer in the body in a subject at any given time. Tumor load can bedetected by e.g., measuring the expression of tumor specific geneticmarkers and measuring tumor size by a number of well-known, biochemicalor imaging methods disclosed herein, infra.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the human bodyof invading pathogens, cells or tissues infected with pathogens,cancerous cells, or, in cases of autoimmunity or pathologicalinflammation, normal human cells or tissues.

As used herein, the term “checkpoint inhibitor sensitive cancer” refersto a cancer that is responsive to checkpoint inhibitors. Administrationof one or more checkpoint inhibitors to patients having such a tumorwould cause a reduction in the tumor load or other desired beneficialclinical outcome related to cancer improvement.

As used herein, the term “effective amount” or “therapeutic amount”refers to an amount of a pharmacological agent effective to treat,eliminate, or mitigate at least one symptom of the disease beingtreated. In some cases, “therapeutically effective amount” or “effectiveamount” can refer to an amount of a functional agent or of apharmaceutical composition useful for exhibiting a detectabletherapeutic or inhibitory effect. The effect can be detected by anyassay method known in the art. The effective amount can be an amounteffective to invoke an antitumor response. The effective amount can bean amount effective to evoke a humoral and/or cellular immune responsein the recipient subject leading to growth inhibition or death of targetcells. For the purpose of this disclosure, the therapeutic amount of thecheckpoint inhibitor is an amount that would reduce tumor load or bringabout other desired beneficial clinical outcomes related to cancerimprovement.

As used herein, the phrase “an amount effective to potentiate” refers tothe amount of of a pharmacological agent effective to enhance theactivity of another therapeutic agent in treating, eliminating, ormitigating at least one symptom of the disease being treated. The agentused to potentiate the activity of another can be effective ornon-effective in treating, eliminating, or mitigating the symptom of thedisease itself. In some cases, the potentiating agent is not effective,and the effect of potentiation can be shown by the increased degree inrelieving the symptom resulting from treatment by the combination of thetwo agents as compared to the treatment with the therapeutic agentalone. In some cases, the potentiating agent itself is effective intreating the symptoms, and the potentiating effect can be shown by asynergistic effect between the potentiating agent and the therapeuticagent. For the purpose of this disclosure, the SGRM acts as apotentiating agent to potentiate the activity of checkpoint inhibitorsin treating cancer, regardless whether the SGRM would be effective intreating the cancer if administered alone. In some embodiments, apotentiating effect of 10% to 1000% can be achieved. In someembodiments, the SGRM is administered at an amount that renders thetumor sensitive to the checkpoint inhibitor, i.e., a showing of areduction of tumor load or other related clinical benefit that would nototherwise appear when the tumor is treated with the checkpoint inhibitorin the absence of the SGRM.

As used herein, the term “combination therapy” refers to theadministration of at least two pharmaceutical agents to a subject totreat a disease. The two agents may be administered simultaneously, orsequentially in any order during the entire or portions of the treatmentperiod. The two agents may be administered following the same ordifferent dosing regimens. In some cases, one agent is administeredfollowing a scheduled regimen while the other agent is administeredintermittently. In some cases, both agents are administeredintermittently. In some embodiments, the one pharmaceutical agent, e.g.,a SGRM, is administered every day, and the other pharmaceutical agent,e.g., a checkpoint inhibitor, is administered weekly or biweekly.

As used herein, the terms “administer,” “administering,” “administered”or “administration” refer to providing a compound or a composition(e.g., one described herein), to a subject or patient.

As used herein, the term “co-administer” refers to administer twocompositions simultaneously or within a short time of each other, e.g.,within about within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours ofeach other.

As used herein, the term “compound” is used to denote a molecular moietyof unique, identifiable chemical structure. A molecular moiety(“compound”) may exist in a free species form, in which it is notassociated with other molecules. A compound may also exist as part of alarger aggregate, in which it is associated with other molecule(s), butnevertheless retains its chemical identity. A solvate, in which themolecular moiety of defined chemical structure (“compound”) isassociated with a molecule(s) of a solvent, is an example of such anassociated form. A hydrate is a solvate in which the associated solventis water. The recitation of a “compound” refers to the molecular moietyitself (of the recited structure), regardless whether it exists in afree form or an associated form.

As used herein, the term “small molecule, non-protein compound” refersto a low molecular weight organic compound, which typically has amolecular weight of less than 900 daltons

As used herein, the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

As used herein, the term “checkpoint protein” refers to a protein thatis present on the surface of certain types of cells, e.g. T cells andcertain tumor cells, and can induce checkpoint signaling pathways andresult in suppression of immune responses. Commonly known checkpointproteins include CTLA4, PD-1, PD-L1, LAG3, B7-H3, B7-H4, CD160, CD244,VISTA, TIGIT, and BMA. (Pardol, 2012, Nature Reviews Cancer 12:252-264;Baksh, 2015, Semin Oncol. 2015 June; 42(3):363-77). Among these, CTLA4,PD-1 and PD-L1 are most well studied and therapies targeting theseproteins are more clinically advanced than therapies targeting othercheckpoint proteins.

As used herein, the term “PD-1” refers to Programmed Cell Death Protein1 (also known as CD279), a cell surface membrane protein of theimmunoglobulin superfamily. PD-1 is expressed by B cells, T cells and NKcells. The major role of PD-1 is to limit the activity of T cells inperipheral tissues during inflammation in response to infection, as wellas to limit autoimmunity. PD-1 expression is induced on activated Tcells and binding of PD-1 to one of its endogenous ligands acts toinhibit T cell activation by inhibiting stimulatory kinases. PD-1 alsoacts to inhibit the TCR “stop signal”. PD-1 is highly expressed on Tregcells (regulatory T cells) and may increase their proliferation in thepresence of ligand (Pardoll, 2012, Nature Reviews Cancer 12:252-264).

As used herein, the term “PD-L1” refers to Programmed Cell Death 1ligand 1 (also known as CD274 and B7-H1), a ligand for PD-1. PD-L1 isfound on activated T cells, B cells, myeloid cells, macrophages, andtumor cells. Although there are two endogenous ligands for PD-1, PD-L1and PD-L2, anti-tumor therapies have focused on anti-PD-L1. The complexof PD-1 and PD-L1 inhibits proliferation of CD8+ T cells and reduces theimmune response (Topalian et al., 2012, N. Engl J. Med. 366:2443-54;Brahmer et al., 2012, N. Engl J. Med. 366:2455-65).

As used herein, the term “CTLA4” refers to Cytotoxic T-lymphocyteantigen 4 (also known as CD152), a member of the immunoglobulinsuperfamily that is expressed exclusively on T cells. CTLA4 acts toinhibit T cell activation and is reported to inhibit helper T cellactivity and enhance regulatory T cell immunosuppressive activity.Although the precise mechanism of action of CTL4-A remains underinvestigation, it has been suggested that it inhibits T cell activationby outcompeting CD28 in binding to CD80 and CD86 on antigen presentingcells, as well as actively delivering inhibitor signals to the T cell(Pardon, 2012; Nature Reviews Cancer 12:252-264).

As used herein, the term “checkpoint inhibitor” refers to any molecules,including antibodies and small molecules, that block theimmunosuppression pathway induced by one or more checkpoint proteins.

As used herein, the term “antibody” as used herein also includes afull-length antibody as well as an “antigen-binding portion” of anantibody. The term “antigen-binding portion”, as used herein, refers toone or more fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., PD-1). Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CHI domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Ed fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL, and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Hustonet al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn etal. 1998, Nature Biotechnology 16: 778). Such single chain antibodiesare also intended to be encompassed within the term “antigen-bindingportion” of an antibody. Any VH and VL sequences of specific scFv can belinked to human immunoglobulin constant region cDNA or genomicsequences, in order to generate expression vectors encoding complete IgGmolecules or other isotypes. VH and VI can also be used in thegeneration of Fab, Fv or other fragments of immunoglobulins using eitherprotein chemistry or recombinant DNA technology. Other forms of singlechain antibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, P., et al.(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al.(1994) Structure 2:1121-1123).

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof, e.g. humanized, chimeric, etc.Antibodies of the invention bind specifically or substantiallyspecifically to one or more checkpoint proteins. The term “monoclonalantibodies” refer to a population of antibody molecules that containonly one species of an antigen binding site capable of immunoreactingwith a particular epitope of an antigen, whereas the term “polyclonalantibodies” and “polyclonal antibody composition” refer to a populationof antibody molecules that contain multiple species of antigen bindingsites capable of interacting with a particular antigen. A monoclonalantibody composition typically displays a single binding affinity for aparticular antigen with which it immunoreacts.

As used herein, the term “antibody effective against a checkpointprotein” refers to an antibody that can bind to the checkpoint proteinand antagonize the checkpoint protein's function in suppressing immuneresponse. For example, an antibody against PD-1 refers to an antibodythat can bind to PD-1 and block the PD-1's inhibitory function on theimmune response, through e.g., blocking the interactions between PD-1and PD-L1. In some cases, an antibody can be against two checkpointproteins, i.e., having the ability of binding to two checkpoint proteinsand inhibiting their function.

As used herein, the term “Glucocorticoid receptor” (“GR”) refers to afamily of intracellular receptors which specifically bind to cortisoland/or cortisol analogs. The glucocorticoid receptor is also referred toas the cortisol receptor. The term includes isoforms of GR, recombinantGR and mutated GR. “Glucocorticoid receptor” (“GR”) refers to the typeII GR which specifically binds to cortisol and/or cortisol analogs suchas dexamethasone (See, e.g., Turner & Muller, J Mol Endocrinol Oct. 1,2005 35 283-292).

“Glucocorticoid receptor modulator” (“GRM”) also known and described inthe scientific and patent literature as a glucocorticoid receptorantagonist refers to any compound which inhibits any biological responseassociated with the binding of GR to an agonist. For example, a GRagonist, such as dexamethasone, increases the activity of tyrosineaminotransferase (TAT) in HepG2 cells (a human liver hepatocellularcarcinoma cell line; ECACC, UK). Accordingly, GR modulators of thepresent invention can be identified by measuring the ability of thecompound to inhibit the effect of dexamethasone. TAT activity can bemeasured as outlined in the literature by A. Ali et al., J. Med. Chem.,2004, 47, 2441-2452. A modulator is a compound with an IC₅₀ (halfmaximal inhibition concentration) of less than 10 micromolar. SeeExample 1, infra.

As used herein, the term “selective glucocorticoid receptor modulator”refers to any composition or compound which inhibits any biologicalresponse associated with the binding of a GR to an agonist. By“selective,” the drug preferentially binds to the GR rather than othernuclear receptors, such as the progesterone receptor (PR), themineralocorticoid receptor (MR) or the androgen receptor (AR). It ispreferred that the selective glucocorticoid receptor antagonist bind GRwith an affinity that is 10× greater ( 1/10^(th) the K_(d) value) thanits affinity to the MR, AR, or PR, both the MR and PR, both the MR andAR, both the AR and PR, or to the MR, AR, and PR. In a more preferredembodiment, the selective glucocorticoid receptor antagonist binds GRwith an affinity that is 100× greater ( 1/100^(th) the K_(d) value) thanits affinity to the MR, AR, or PR, both the MR and PR, both the MR andAR, both the AR and PR, or to the MR, AR, and PR. In another embodiment,the selective glucocorticoid receptor antagonist binds GR with anaffinity that is 1000× greater ( 1/1000^(th) the K_(d) value) than itsaffinity to the MR, AR, or PR, both the MR and PR, both the MR and AR,both the AR and PR, or to the MR, AR, and PR.

As used herein, the term “composition” is intended to encompass aproduct comprising the specified ingredients such as the said compounds,their tautomeric forms, their derivatives, their analogues, theirstereoisomers, their polymorphs, their pharmaceutically acceptablesalts, esters, ethers, metabolites, mixtures of isomers, theirpharmaceutically acceptable solvates and pharmaceutically acceptablecompositions in specified amounts, as well as any product which results,directly or indirectly, from combination of the specified ingredients inthe specified amounts. Such term in relation to a pharmaceuticalcomposition is intended to encompass a product comprising the activeingredient (s), and the inert ingredient (s) that make up the carrier,as well as any product which results, directly or indirectly, incombination, complexation or aggregation of any two or more of theingredients, or from dissociation of one or more of the ingredients, orfrom other types of reactions or interactions of one or more of theingredients. Accordingly, the pharmaceutical compositions of the presentinvention are meant to encompass any composition made by admixingcompounds of the present invention and their pharmaceutically acceptablecarriers.

As used herein, the phrase “not otherwise indicated for treatment with aglucocorticoid receptor modulator” refers to refers to a patient that isnot suffering from any condition recognized by the medical community tobe effectively treatable with glucocorticoid receptor antagonists, withthe exception of hepatic steatosis. Conditions known in the art andaccepted by the medical community to be effectively treatable withglucocorticoid receptor antagonists include: psychosis associated withinterferon-α therapy, psychotic major depression, dementia, stressdisorders, autoimmune disease, neural injuries, and Cushing's syndrome,

In some embodiments, the term “consisting essentially of” refers to acomposition in a formulation whose only active ingredient is theindicated active ingredient, however, other compounds may be includedwhich are for stabilizing, preserving, etc. the formulation, but are notinvolved directly in the therapeutic effect of the indicated activeingredient. In some embodiments, the term “consisting essentially of”can refer to compositions which contain the active ingredient andcomponents which facilitate the release of the active ingredient. Forexample, the composition can contain one or more components that provideextended release of the active ingredient over time to the subject. Insome embodiments, the term “consisting” refers to a composition, whichcontains the active ingredient and a pharmaceutically acceptable carrieror excipient.

The term “steroidal backbone” in the context of glucocorticoid receptorantagonists containing such refers to glucocorticoid receptorantagonists that contain modifications of the basic structure ofcortisol, an endogenous steroidal glucocorticoid receptor ligand. Thebasic structure of a steroidal backbone is provided as Formula I:

The two most commonly known classes of structural modifications of thecortisol steroid backbone to create glucocorticoid antagonists includemodifications of the 11-β hydroxy group and modification of the 17-βside chain (See, e.g., Lefebvre (1989) J. Steroid Biochem. 33: 557-563).

As used herein, the phrase “non-steroidal backbone” in the context ofSGRMs refers to SGRMs that do not share structural homology to, or arenot modifications of, cortisol with its steroid backbone containingseventeen carbon atoms, bonded in four fused rings. Such compoundsinclude synthetic mimetics and analogs of proteins, including partiallypeptidic, pseudopeptidic and non-peptidic molecular entities.

Non-steroidal SGRM compounds include SGRMs having a fused azadecalinbackbone, a heteroaryl ketone fused azadecalin backbone, and anoctahydro fused azadecalin backbone. Exemplary glucocorticoid receptormodulators having a fused azadecalin backbone include those described inU.S. Pat. Nos. 7,928,237 and 8,461,172. Exemplary glucocorticoidreceptor modulators having a heteroaryl ketone fused azadecalin backboneinclude those described in U.S. 2014/0038926, Exemplary glucocorticoidreceptor modulators having an octahydro fused azadecalin backboneinclude those described in U.S. Provisional Patent Appl. No. 61/908,333,entitled Octahydro Fused Azadecalin Glucocorticoid Receptor Modulators,filed on Nov. 25, 2013.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. Alkyl can include anynumber of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆, andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec butyl, tert butyl,pentyl, isopentyl, and hexyl.

“Alkoxy” refers to an alkyl group having an oxygen atom that connectsthe alkyl group to the point of attachment: alkyl-O—. As for the alkylgroup, alkoxy groups can have any suitable number of carbon atoms, suchas C₁₋₆. Alkoxy groups include, for example, methoxy, ethoxy, propoxy,iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,pentoxy, hexoxy, etc.

“Halogen” refers to fluorine, chlorine, bromine, and iodine.

“Haloalkyl” refers to alkyl, as defined above, where some or all of thehydrogen atoms are replaced with halogen atoms. As for the alkyl group,haloalkyl groups can have any suitable number of carbon atoms, such asC₁₋₆, and include trifluoromethyl, fluoromethyl, etc.

The term “perfluoro” can be used to define a compound or radical whereall the hydrogens are replaced with fluorine. For example,perfluoromethane includes 1,1,1-trifluoromethyl.

“Haloalkoxy” refers to an alkoxy group where some or all of the hydrogenatoms are substituted with halogen atoms. As for the alkyl group,haloalkoxy groups can have any suitable number of carbon atoms, such asC₁₋₆. The alkoxy groups can be substituted with 1, 2, 3, or morehalogens. When all the hydrogens are replaced with a halogen, forexample by fluorine, the compounds are per-substituted, for example,perfluorinated. Haloalkoxy includes, but is not limited to,trifluoromethoxy, 2,2,2-trifluoroethoxy, and perfluoroethoxy.

“Cycloalkyl” refers to a saturated or partially unsaturated, monocyclic,fused bicyclic, or bridged polycyclic ring assembly containing from 3 to12 ring atoms, or the number of atoms indicated. Cycloalkyl can includeany number of carbons, such as C₃₋₆, C₄₋₆, C₅₋₆, C₃₋₈, C₄₋₈, —C₅₋₈,C₆₋₈, C₃₋₉, C₃₋₁₀, C₃₋₁₁, and C₃₋₁₂. Saturated monocyclic cycloalkylrings include, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cyclooctyl. Saturated bicyclic and polycyclic cycloalkylrings include, for example, norbornene, [2.2.2] bicyclooctane,decahydronaphthalene, and adamantane. Cycloalkyl groups can also bepartially unsaturated, having one or more double or triple bonds in thering. Representative cycloalkyl groups that are partially unsaturatedinclude, but are not limited to, cyclobutene, cyclopentene, cyclohexene,cyclohexadiene (1,3- and 1,4-isomers), cycloheptene, cycloheptadiene,cyclooctene, cyclooctadiene (1,3-, 1,4- and 1,5-isomers), norbornene,and norbornadiene. When cycloalkyl is a saturated monocyclic C₃₋₈exemplary groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Whencycloalkyl is a saturated monocyclic C₃₋₆ cycloalkyl, exemplary groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,and cyclohexyl.

“Heterocycloalkyl” refers to a saturated ring system having from 3 to 12ring members and from 1 to 4 heteroatoms of N, O, and S. Additionalheteroatoms can also be useful, including but not limited to, B, Al, Si,and P. The heteroatoms can also be oxidized, such as, but not limitedto, —S(O)— and —S(O)². Heterocycloalkyl groups can include any number ofring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5 to 8, 6 to8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Any suitablenumber of heteroatoms can be included in the heterocycloalkyl groups,such as 1, 2, 3, or 4, or 1 to 2, 1 to 3, 1 to 4, 2 to 3, 2 to 4, or 3to 4. The heterocycloalkyl group can include groups such as aziridine,azetidine, pyrrolidine, piperidine, azepane, azocane, quinuclidine,pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and 1,4-isomers),oxirane, oxetane, tetrahydrofuran, oxane (tetrahydropyran), oxepane,thiirane, thietane, thiolane (tetrahydrothiophene), thiane(tetrahydrothiopyran), oxazolidine, isoxalidine, thiazolidine,isothiazolidine, dioxolane, dithiolane, morpholine, thiomorpholine,dioxane, or dithiane. The heterocycloalkyl groups can also be fused toaromatic or non-aromatic ring systems to form members including, but notlimited to, indoline.

When heterocycloalkyl includes 3 to 8 ring members and 1 to 3heteroatoms, representative members include, but are not limited to,pyrrolidine, piperidine, tetrahydrofuran, oxane, tetrahydrothiophene,thiane, pyrazolidine, imidazolidine, piperazine, oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, morpholine,thiomorpholine, dioxane and dithiane. Heterocycloalkyl can also form aring having 5 to 6 ring members and 1 to 2 heteroatoms, withrepresentative members including, but not limited to, pyrrolidine,piperidine, tetrahydrofuran, tetrahydrothiophene, pyrazolidine,imidazolidine, piperazine, oxazolidine, isoxazolidine, thiazolidine,isothiazolidine, and morpholine.

“Aryl” refers to an aromatic ring system having any suitable number ofring atoms and any suitable number of rings. Aryl groups can include anysuitable number of ring atoms, such as 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 ring atoms, as well as from 6 to 10, 6 to 12, or 6 to 14 ringmembers. Aryl groups can be monocyclic, fused to form bicyclic ortricyclic groups, or linked by a bond to form a biaryl group.Representative aryl groups include phenyl, naphthyl and biphenyl. Otheraryl groups include benzyl, that has a methylene linking group. Somearyl groups have from 6 to 12 ring members, such as phenyl, naphthyl, orbiphenyl. Other aryl groups have from 6 to 10 ring members, such asphenyl or naphthyl. Some other aryl groups have 6 ring members, such asphenyl. Aryl groups can be substituted or unsubstituted.

“Heteroaryl” refers to a monocyclic, fused bicyclic, or tricyclicaromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 5of the ring atoms are a heteroatom such as N, O, or S. Additionalheteroatoms can also be useful, including but not limited to, B, Al, Si,and P. The heteroatoms can also be oxidized, such as, but not limitedto, N-oxide, —S(O)—, and —S(O)₂—. Heteroaryl groups can include anynumber of ring atoms, such as 3 to 6, 4 to 6, 5 to 6, 3 to 8, 4 to 8, 5to 8, 6 to 8, 3 to 9, 3 to 10, 3 to 11, or 3 to 12 ring members. Anysuitable number of heteroatoms can be included in the heteroaryl groups,such as 1, 2, 3, 4, or 5; or 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2to 4, 2 to 5, 3 to 4, or 3 to 5. Heteroaryl groups can have from 5 to 8ring members and from 1 to 4 heteroatoms, or from 5 to 8 ring membersand from 1 to 3 heteroatoms, or from 5 to 6 ring members and from 1 to 4heteroatoms, or from 5 to 6 ring members and from 1 to 3 heteroatoms.The heteroaryl group can include groups such as pyrrole, pyridine,imidazole, pyrazole, triazole, tetrazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4-, and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, and isoxazole. The heteroarylgroups can also be fused to aromatic ring systems, such as a phenylring, to form members including, but not limited to, benzopyrroles suchas indole and isoindole, benzopyridines such as quinoline andisoquinoline, benzopyrazine (quinoxaline), benzopyrimidine(quinazoline), benzopyridazines such as phthalazine and cinnoline,benzothiophene, and benzofuran. Other heteroaryl groups includeheteroaryl rings linked by a bond, such as bipyridine. Heteroaryl groupscan be substituted or unsubstituted.

The heteroaryl groups can be linked via any position on the ring. Forexample, pyrrole includes 1-, 2-, and 3-pyrrole; pyridine includes 2-,3- and 4-pyridine; imidazole includes 1-, 2-, 4- and 5-imidazole;pyrazole includes 1-, 3-, 4- and 5-pyrazole; triazole includes 1-, 4-and 5-triazole; tetrazole includes 1- and 5-tetrazole; pyrimidineincludes 2-, 4-, 5- and 6-pyrimidine; pyridazine includes 3- and4-pyridazine; 1,2,3-triazine includes 4- and 5-triazine; 1,2,4-triazineincludes 3-, 5- and 6-triazine; 1,3,5-triazine includes 2-triazine;thiophene includes 2- and 3-thiophene; furan includes 2- and 3-furan;thiazole includes 2-, 4- and 5-thiazole; isothiazole includes 3-, 4- and5-isothiazole; oxazole includes 2-, 4- and 5-oxazole; isoxazole includes3-, 4- and 5-isoxazole; indole includes 1-, 2- and 3-indole; isoindoleincludes 1- and 2-isoindole; quinoline includes 2-, 3- and 4-quinoline;isoquinoline includes 1-, 3- and 4-isoquinoline; quinazoline includes 2-and 4-quinoazoline; cinnoline includes 3- and 4-cinnoline;benzothiophene includes 2- and 3-benzothiophene; and benzofuran includes2- and 3-benzofuran.

Some heteroaryl groups include those having from 5 to 10 ring membersand from 1 to 3 ring atoms including N, O, or S, such as pyrrole,pyridine, imidazole, pyrazole, triazole, pyrazine, pyrimidine,pyridazine, triazine (1,2,3-, 1,2,4- and 1,3,5-isomers), thiophene,furan, thiazole, isothiazole, oxazole, isoxazole, indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, and benzofuran. Other heteroaryl groupsinclude those having from 5 to 8 ring members and from 1 to 3heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole, triazole,pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole, andisoxazole. Some other heteroaryl groups include those having from 9 to12 ring members and from 1 to 3 heteroatoms, such as indole, isoindole,quinoline, isoquinoline, quinoxaline, quinazoline, phthalazine,cinnoline, benzothiophene, benzofuran and bipyridine. Still otherheteroaryl groups include those having from 5 to 6 ring members and from1 to 2 ring heteroatoms including N, O or S, such as pyrrole, pyridine,imidazole, pyrazole, pyrazine, pyrimidine, pyridazine, thiophene, furan,thiazole, isothiazole, oxazole, and isoxazole.

Some heteroaryl groups include from 5 to 10 ring members and onlynitrogen heteroatoms, such as pyrrole, pyridine, imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), indole, isoindole, quinoline, isoquinoline, quinoxaline,quinazoline, phthalazine, and cinnoline. Other heteroaryl groups includefrom 5 to 10 ring members and only oxygen heteroatoms, such as furan andbenzofuran. Some other heteroaryl groups include from 5 to 10 ringmembers and only sulfur heteroatoms, such as thiophene andbenzothiophene. Still other heteroaryl groups include from 5 to 10 ringmembers and at least two heteroatoms, such as imidazole, pyrazole,triazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4- and1,3,5-isomers), thiazole, isothiazole, oxazole, isoxazole, quinoxaline,quinazoline, phthalazine, and cinnoline.

“Heteroatoms” refers to O, S, or N.

“Salt” refers to acid or base salts of the compounds used in the methodsof the present invention. Illustrative examples ofpharmaceutically-acceptable salts are mineral acid (hydrochloric acid,hydrobromic acid, phosphoric acid, and the like) salts, organic acid(acetic acid, propionic acid, glutamic acid, citric acid, and the like)salts, and quaternary ammonium (methyl iodide, ethyl iodide, and thelike) salts. It is understood that the pharmaceutically-acceptable saltsare non-toxic. Additional information on suitablepharmaceutically-acceptable salts can be found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, which is incorporated herein by reference.

“Isomers” refers to compounds with the same chemical formula but whichare structurally distinguishable.

“Tautomer” refers to one of two or more structural isomers which existin equilibrium and which are readily converted from one form to another.

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to produce compounds which are notinherently unstable—and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions—such asaqueous, neutral, or physiological conditions.

“Pharmaceutically-acceptable excipient” and “pharmaceutically-acceptablecarrier” refer to a substance that aids the administration of an activeagent to—and absorption by—a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically-acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors and colors, and the like. One of ordinary skill in the art willrecognize that other pharmaceutical excipients are useful in the presentinvention.

As used herein, the term “GR⁺ cancer” refers to a cancer that expressesGR. GR expression can be determined by routine molecular and biochemicalmethods in the art, for example, immunohistochemistry staining. In oneembodiment, a GR⁺ cancer is one that has at least 10% tumor cellsshowing nuclear staining of GR at any intensity. In another embodiment,a GR⁺ cancer is one that has a H-score, using methods disclosed insection “identifying GR expression”, equal or greater than apredetermined threshold, e.g., 150.

C. Select Patient Population

i. Diagnosing Cancer

Cancers are characterized by uncontrolled growth and/or spread ofabnormal cells. A biopsy is tyically taken and the cell or tissue fromthe biopsy is examined under a microscope in order to confirm asuspected condition. In some cases, additional tests need to beperformed on the cells' proteins, DNA, and RNA to verify the diagnosis.

ii. Identifying Checkpoint Inhibitor Sensitive Cancer

In some embodiments of the invention, methods are used to treat patientshaving at least one checkpoint inhibitor sensitive cancer. Checkpointinhibitor sensitive cancers are those that are responsive to checkpointinhibitors, i.e., administration of one or more checkpoint inhibitorscan reduce tumor load or achieve beneficial or desired clinical resultsrelated to cancer improvement. For example, the administration of thecheckpoint inhibitor may bring about one or more of the following:reducing the number of cancer cells; reducing the tumor size; inhibiting(i.e., slowing to some extent and/or stop) cancer cell infiltration intoperipheral organs; inhibiting (i.e., slowing to some extent and/or stop)tumor metastasis; inhibiting, to some extent, tumor growth; and/orrelieving to some extent one or more of the symptoms associated with thedisorder; shrinking the size of the tumor; decreasing symptoms resultingfrom the disease; increasing the quality of life of those suffering fromthe disease; decreasing the dose of other medications required to treatthe disease; delaying the progression of the disease; and/or prolongingsurvival of patients.

Checkpoint inhibitor sensitive tumors often have high expression ofligands, e.g., PD-L1 or B7, that bind to checkpoint proteins, PD-1 orCTLA-4, respectively. These interactions suppress immune responsesagainst the tumor cells. Non-limiting examples of checkpoint inhibitorsensitive tumors include lung cancer, liver cancer, ovarian cancer,cervical cancer, skin cancer, bladder cancer, colon cancer, breastcancer, glioma, renal carcinoma, stomach cancer, esophageal cancer, oralsquamous cell cancer, head/neck cancer, melanoma, sarcoma, renal celltumor, hepatocellular tumor, glioblastoma, neuroendocrine tumor, bladdercancer, pancreatic cancer, gall bladder cancer, gastric cancer, prostatecancer, endometrial cancer, thyroid cancer and mesothelioma.

iii. Identifying GR Expression

In some embodiments, the checkpoint inhibitor sensitive cancer is also aGR⁺ cancer. GR expression in cancer cells can be examined by using oneor more of the routine biochemical analyses. In some embodiments, GRexpression is determined by detecting GR transcript expression, usingmethods such as microarray and RT-PCR. In other embodiments, GRexpression is determined by detecting protein expression, using methodssuch as, western blot analysis and immunohistochemistry staining. In yetother embodiments, the GR expression is determined using a combinationof these methods.

In a preferred embodiment, immunohistochemistry staining is performedand a H-score method is used to quantify the expression of GR on cancertissues. In one exemplar assay, Formalin-fixed, paraffin-embedded tumortissue sections are deparaffinized and treated with antigen retrievalsolution to render the glucocorticoid receptors readily accessible toanti-GR antibodies. Anti-GR antibodies are then incubated with thetissue sections and the antibodies bound to the GR on the tissuesections are detected by addition of a horse peroxidase (HRP) conjugatedsecondary antibody that recognizes the anti-GR antibody. The HRP on thesecondary antibody conjugate catalyzes a colorimetric reaction and uponcontacting the appropriate substrate, produces a staining in thelocations where GR is present. In one approach, the intensity level ofthe GR staining is represented by 0 for negative staining. 1+ for weakstaining, 2+ for moderate staining, and 3+ for strong staining. Seewww.ihcworld.com/ihc_scoring.htm. The percentage of GR⁺ cells of eachintensity level is multiplied with the intensity level, and the resultsfor all intensity levels are summed to generate a H-score between 0-300.In one embodiment, the cancer type having a H-score equal to or higherthan a predetermined threshold is considered GR⁺ cancer. In a preferredembodiment, the threshold is 150. In another embodiment, a GR⁺ cancer isone that has at least 10% tumor cells showing GR staining at anyintensity. A number of cancer types are GR⁺, using the threshold ofH-score 150. See Table 1, below. A majority of these cancer types arealso checkpoint inhibitor sensitive cancers as shown by publishedresults of clinical trials. See, www.clinicaltrial.gov.

D. Checkpoint Inhibitors

The method disclosed herein uses at least one SGRM in combination withat least one checkpoint inhibitor to treat cancers. In some embodiments,the checkpoint inhibitor is an antibody (“CIA”) against at least onecheckpoint protein. In some embodiments, the checkpoint inhibitor is asmall molecule, non-protein compound (“CIC”) that blocks theimmunosuppression pathway induced by one or more checkpoint proteins.

i. Checkpoint Inhibitor Antibodies (“CIA”)

In one embodiment, the method for treating cancer comprisesadministering a SGRM in combination with a checkpoint inhibitorantibody. Such an antibody can block the immunosuppression activity ofthe checkpoint protein. A number of such antibodies have already beenshown to be effective in treating cancers, e.g., antibodies againstPD-1, CTLA4, and PD-L1.

Anti-PD 1 antibodies have been used for the treatment of melanoma,non-small-cell lung cancer, bladder cancer, prostate cancer, colorectalcancer, head and neck cancer, triple-negative breast cancer, leukemia,lymphoma and renal cell cancer. Exemplary anti-PD-1 antibodies includelambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERSSQUIBB), AMP-224 (MERCK), and pidilizumab (CT-011, CURETECH LTD.).

Anti-PD-L1 antibodies have been used for treatment of non-small celllung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreaticcancer, gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies. Exemplary anti-PD-L1 antibodies include MDX-1105(MEDAREX), MEDI4736 (MEDIMMUNE), MPDL3280A (GENENTECH) and BMS-936559(BRISTOL-MYERS SQUIBB).

Anti-CTLA4 antibodies have been used in clinical trials for thetreatment of melanoma, prostate cancer, small cell lung cancer,non-small cell lung cancer. A significant feature of anti-CTL4A is thekinetics of anti-tumor effect, with a lag period of up to 6 months afterinitial treatment required for physiologic response. In some cases,tumors may actually increase in size after treatment initiation, beforea reduction is seen (Pardoll, 2012, Nature Reviews Cancer 12:252-264).Exemplary anti-CTLA4 CIAs include ipilimumab (Bristol-Myers Squibb) andtremelimumab (PFIZER).

CIAs against other checkpoint proteins, such as LAG3, B7-H3, B7-H4 andTIM3, may also be used in combination with the SGRMs disclosed herein totreat cancers.

The CIAs used in this disclosure can be a combination of different CIAs,especially if the target checkpoint proteins, e.g., PD-1 and CTLA4,suppress immune response via different signaling pathways. Thus acombination of CIAs against either of the checkpoint proteins or asingle CIA that is against both checkpoint proteins may provide anenhanced immune response.

Generating CIAs

CIAs can be developed using methods well known in the art. See, forexample, Kohler and Milstein, Nature 256: 495 (1975), and Coligan et al.(eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (JohnWiley & Sons 1991). Monoclonal antibodies can be obtained by injectingmice with a composition comprising an antigen, e.g. a checkpoint proteinor an epitope of thereof, removing the spleen to obtain B-lymphocytes,fusing the B-lymphocytes with myeloma cells to produce hybridomas,cloning the hybridomas, selecting positive clones which produceantibodies to the antigen, culturing the clones that produce antibodiesto the antigen, and isolating the antibodies from the hybridomacultures.

Monoclonal antibodies produced can be isolated and purified fromhybridoma cultures by a variety of well-established techniques. Suchisolation techniques include affinity chromatography with Protein-ASepharose, size-exclusion chromatography, and ion-exchangechromatography. See, for example, Coligan at pages 27.1-2.7.12 and pages2.9.1-2.9.3. Also, see Baines et al., “Purification of Immunoglobulin G(IgG),” in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (TheHumana Press, Inc. 1992). After the initial raising of antibodies to acheckpoint protein, the antibodies can be sequenced and subsequentlyprepared by recombinant techniques. Humanization and chimerization ofmurine antibodies and antibody fragments are well known to those skilledin the art. See, for example, Leung et al. Hybridoma 13:469 (1994);US20140099254 A1.

Human antibodies can be produced using transgenic mice that have beengenetically engineered to produce specific human antibodies in responseto antigenic challenge using a checkpoint protein. See Green et al.,Nature Genet. 7: 13 (1994), Lonberg et al., Nature 368:856 (1994). Humanantibodies against a checkpoint protein also can be constructed bygenetic or chromosomal trandfection methods, phage display technology,or by in vitro activated B cells. See e.g., McCafferty et al., 1990,Nature 348: 552-553; U.S. Pat. Nos. 5,567,610 and 5,229,275.

Modifyin CIAs

CIAs may also be produced by introducing conservative modificationsrelative to the existing CIAs. For example, a modifed CIA may compriseheavy and light chain variable regions, and/or a Fc region that arehomologous to the counterparts of an antibody produced above. Themodified CIA that can be used for the method disclosed herein mustretain the desired functional properties of being able to block thecheckpoint signaling pathway.

CIAs may also be produced by altering protein modification sites. Forexample, sites of glycosylation of the antibody can be altered toproduce an antibody lacking glycosylation and the so modified CIAstypically have increased affinity of the antibody for antigen.Antibodies can also be pegylated by reacting with polyethylene glycol(PEG) under conditions in which one or more PEG groups become attachedto the antibody. Pegylation can increase the biological half-life of theantibody. Antibodies having such modifications can also be used incombination with the selective GR modulator disclosed herein so long asit retains the desired functional properties of blocking the checkpointpathways.

ii. Small Molecule, Non-Protein Checkpoint Inhibitor Compounds (“CICs”)

In another embodiment, the method for treating cancer, e.g. a checkpointinhibitor sensitive cancer, uses a SGRM in combination with a CIC. A CICis a small molecule, non-protein compound that antagonizes a checkpointprotein's immune suppression function. Many CICs are known in the art,for example, those disclosed in PCT publications WO2015034820,WO20130144704, and WO2011082400.

CICs can also be identified using any of the numerous approaches incombinatorial library methods known in the art and disclosed in, e.g.,European patent application EP2360254. The cominatorial librariesinclude: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145).

iii. Evaluating the Functional Properties of the Candidate CheckpointInhibitors

A number of well-known assays can be used to assess whether a candidate,i.e., an antibody generated by immunizing an animal with an antigencomprising a checkpoint protein, an epitope of the checkpoint protein,or a test compound from combinatorial libraries, as disclosed above, isa checkpoint inhibitor. Non-limiting exemplar assays include bindingassays—such as Enzyme-Linked Immunosorbent Assays (ELISAs),radioimmunoassays (RIA)—, Fluorescence-Activated Cell Sorting (FACS)analysis, cell-based assays, and in vivo assays.

Binding Assays

In one embodiment, the assay is a direct binding assay. The checkpointprotein can be coupled with a radioisotope or enzymatic label such thatbinding of the checkpoint protein and the candidate can be determined bydetecting the labeled checkpoint protein in a complex. For example, acheckpoint protein can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, eitherdirectly or indirectly, and the radioisotope detected by direct countingof radio-emission or by scintillation counting. Determining the abilityof candidates to bind their cognate checkpoint protein can beaccomplished, e.g., by measuring direct binding. Alternatively,checkpoint protein molecules can be enzymatically labeled with, forexample, horseradish peroxidase, alkaline phosphatase, or luciferase,and binding of the candidates to the target checkpoint protein isdetermined by conversion of an appropriate substrate to product.

Enzyme-linked immunosorbent assay (ELISA) are commonly used to evaluatea CIA candidate's binding specificity to its target checkpoint protein.In a typical assay, microtiter plates are coated with the checkpointprotein by coating overnight at 37° C. with 5 μg/ml checkpoint protein.Serum samples comprising candidate CIAs are diluted in PBS, 5% serum,0.5% Tween-20 and are incubated in wells for 1 hour at room temperature,followed by the addition of anti-human IgG Fc and IgG F(ab′)-horseradishperoxidase in the same diluent. After 1 hour at room temperature enzymeactivity is assessed by addition of ABTS substrate (Sigma, St. LouisMo.) and read after 30 minutes at 415-490 nm.

The binding kinetics (e.g., binding affinity) of the candidates also canbe assessed by standard assays known in the art, such as by Biacoreanalysis (Biacore AB, Uppsala, Sweden). In one exemplary assay, apurified recombinant human checkpoint protein is covalently linked to aCM5 chip (carboxy methyl dextran coated chip) via primary amines, usingstandard amine coupling chemistry and kit provided by Biacore. Bindingis measured by flowing the candidates in HBS EP buffer (provided byBiacore AB) at a concentration of 267 nM at a flow rate of 50 μl/min.The checkpoint protein-candidate association kinetics are followed for 3minutes and the dissociation kinetics are followed for 7 minutes. Theassociation and dissociation curves are fitted to a 1:1 Langmuir bindingmodel using BIA evaluation software (Biacore AB). To minimize theeffects of avidity in the estimation of the binding constants, only theinitial segment of data corresponding to association and dissociationphases are used for fitting. The K^(D), K_(on) and K_(off) values of theinteraction can be measured. Preferred checkpoint inhibitors can bind totheir target checkpoint protein with a Kd of 1×10⁻⁷M or less

For checkpoint proteins that block immune responses through binding to aligand, additional binding assays may be employed to test for theability of the candidate to block binding of the ligands to thecheckpoint protein. In one exemplary assay, flow cytometry is used totest the blocking of the binding of the ligand (e.g., PD-L1) to thecheckpoint protein (e.g., PD-1) expressed on transfected CHO cells.Various concentrations of the candidate are added to the suspension ofcells expressing the checkpoint protein and incubated at 4° C. for 30minutes. Unbound inhibitor is washed off and FITC-labeled ligand proteinis added into the tubes and incubated at 4° C. for 30 minutes. FACSanalysis is performed using a FACScan flow cytometer (Becton Dickinson.San Jose, Calif.). The mean fluorescent intensity (MFI) of staining ofthe cells indicates the amount of ligand that is bound to the checkpointproteins. A reduced MFI in the sample to which the candidate is addedindicates that the candidate is effective in blocking the binding of theligand to the target checkpoint protein.

Homogenous Time-Resolved Fluorescence (HTRF) binding assay, such asdescribed in PCT publication WO2015034820, can also be used to assay thecandidate's ability to block the checkpoint protein-ligand interaction.In one embodiment, the CICs used in the method can inhibit thePD-1/PD-L1 interaction with IC₅₀ values of 10 pM or less, for example,from 0.01 to 10 pM, preferrably, 1 pM or less, e.g., from 0.01 to 1 pM,as measured by the PD-1/PD-L1 Homogenous Time-Resolved Fluorescence(HTRF) binding assay.

Cell Based Assays

In another embodiment, the assay to evaluate whether a candidate is acheckpoint inhibitor is a cell based assay. The Mixed LymphocyteReaction (MLR) assay, as described in U.S. Pat. No. 8,008,449, isroutinely used to measure T cell proliferation, production of IL-2and/or IFN-γ. In one exemplary assay, human T cells are purified fromPBMCs using a human CD4⁺ T cell enrichment column (R&D systems). Acandidate is added to a number of T cell cultures at differentconcentrations. The cells are cultured for 5 days at 37° C. and 100 μlof medium is taken from each culture for cytokine measurement. Thelevels of IFN-gamma and other cytokines are measured using OptEIA ELISAkits (BD Biosciences). The cells are labeled with ³H-thymidine, culturedfor another 18 hours, and analyzed for cell proliferation. Resultsshowing that, as compared to control, the culture containing thecandidate shows increased T cell proliferation, increased production ofIL-2, and/or IFN-gamma indicate the candidate is effective in blockingcheckpoint protein's inhibition of T cell immune response.

In Vivo Assays

In another embodiment, the assay used to evaluate whether a candidate isa checkpoint inhibitor is a in vivo assay. In one exemplary assay,female AJ mice between 6-8 weeks of age (Harlan Laboratories) arerandomized by weight into 6 groups. The mice are implantedsubcutaneously in the right flank with 2×10⁶ SA1/N fibrosarcoma cellsdissolved in 200 μl of DMEM media on day 0. The mice are treated withPBS vehicle, or the candidate at a predetermined dosage. The animals aredosed by intraperitoneal injection with approximately 200 μl of PBScontaining the candidate or vehicle on days 1, 4, 8 and 11. The mice aremonitored twice weekly for tumor growth for approximately 6 weeks. Usingan electronic caliper, the tumors are measured three dimensionally(height×width×length) and tumor volume is calculated. Mice areeuthanized when the tumors reach tumor end point (1500 mm³) or the miceshow greater than 15% weight loss. A result showing that a slower tumorgrowth in the candidate treated group as compared to controls, or alonger mean time to reach the tumor end point volume (1500 mm³) is anindication that the candidate has activity in inhibiting cancer growth.

E. Glucocorticoid Receptor Modulators (GRM)

The combination therapy for treating cancer disclosed herein alsoinvolves at least one selective glucocorticoid receptor modulator incombination with at least one checkpoint inhibitor to treat a cancer,e.g., a checkpoint inhibitor sensitive cancer. Provided herein, areclasses of exemplary GRMs and specific members of such classes. However,one of skill in the art will readily recognize other related orunrelated SGRMs that can be employed in the treatment methods describedherein.

1. GRMs Having a Steroidal Backbone

In some embodiments, an effective amount of a SGRM with a steroidalbackbone is administered to a subject for cancer treatment. SteroidalGRMs can be obtained by modification of the basic structure ofglucocorticoid agonists, i.e., varied forms of the steroid backbone. Thestructure of cortisol can be modified in a variety of ways. The two mostcommonly known classes of structural modifications of the cortisolsteroid backbone to create GRAs include modifications of the 11-βhydroxy group and modification of the 17-β side chain (See, e.g.,Lefebvre, J. Steroid Biochem. 33:557-563, 1989).

Examples of steroidal GR antagonists include androgen-type steroidalcompounds as described in U.S. Pat. No. 5,929,058, and the compoundsdisclosed in U.S. Pat. Nos, 4,296,206; 4,386,085; 4,447,424; 4,477,445;4,519,946; 4,540,686; 4,547,493; 4,634,695; 4,634,696; 4,753,932;4,774,236; 4,808,710; 4,814,327; 4,829,060; 4,861,763; 4,912,097;4,921,638; 4,943,566; 4,954,490; 4,978,657; 5,006,518; 5,043,332;5,064,822; 5,073,548; 5,089,488; 5,089,635; 5,093,507; 5,095,010;5,095,129; 5,132,299; 5,166,146; 5,166,199; 5,173,405; 5,276,023;5,380,839; 5,348,729; 5,426,102; 5,439,913; 5,616,458, 5,696,127, and6,303,591. Such steroidal GR antagonists include cortexolone,dexamethasone-oxetanone, 19-nordeoxycorticosterone, 19-norprogesterone,cortisol-21-mesylate; dexamethasone-21-mesylate,11β-(4-dimethylaminoethoxyphenyl)-17α-propynyl-17β-hydroxy-4,9-estradien-3-one(RU009), and(17α)-17-hydroxy-19-(4-methylphenyl)androsta-4,9(11)-dien-3-one (RU044).

Other examples of steroidal antiglucocorticoids are disclosed in VanKampen et al. (2002) Eur. J. Pharmacol. 457(2-3):207, WO 03/043640, EP 0683 172 B1, and EP 0 763 541 BL each of which is incorporated herein byreference. EP 0 763 541 B1 and Hoyberg et al., Int'l J. ofNeuro-psychopharmacology, 5:Supp. 1, S148 (2002) disclose the compound(11β,17β)-11-(1,3-benzodioxo-5-yl)-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one(ORG 34517), which in one embodiment, is administered in an amounteffective to treat an ACTH-secreting tumor in a subject.

2. Removal or Substitution of the 11-β Hydroxy Group

Glucocorticoid antagonists with modified steroidal backbones comprisingremoval or substitution of the 11-β hydroxy group are administered inone embodiment of the invention. This class includes natural GRMs,including cortexolone, progesterone and testosterone derivatives, andsynthetic compositions, such as mifepristone (Lefebvre, et al. supra).Preferred embodiments of the invention include all 11-β aryl steroidbackbone derivatives because, in some cases, these compounds can bedevoid of progesterone receptor (PR) binding activity (Agarwal, FEBS217:221-226, 1987). In another embodiment an 11-β phenyl-aminodimethylsteroid backbone derivative, which is both an effectiveanti-glucocorticoid and anti-progesterone agent, is administered. Thesecompositions can act as reversibly-binding steroid receptor antagonists.For example, when bound to a 11-β phenyl-aminodimethyl steroid, thesteroid receptor can be maintained in a conformation that cannot bindits natural ligand, such as cortisol in the case of GR (Cadepond, 1997,supra).

Synthetic 11-beta phenyl-aminodimethyl steroids include mifepristone,also known as RU486, or17-β-hydrox-11-β-(4-dimethyl-aminophenyl)17-α-(1-propynyl)estra-4,9-dien-3-one).Mifepristone has been shown to be a powerful antagonist of both theprogesterone and glucocorticoid (GR) receptors. Thus, in someembodiments, the GRM administered to treat an ACTH-secreting tumor ismifepristone, or a salt, tautomer, or derivative thereof. In otherembodiments, however, administration of mifepristone is specificallyexcluded as a GRM for treatment of an ACTH-secreting tumor.

Another 11-β phenyl-aminodimethyl steroid shown to have GR antagonisteffects includes the dimethyl aminoethoxyphenyl derivative RU009(RU39.009),11-β-(4-dimethyl-aminoethoxyphenyl)-17-α-(propynyl-17-β-hydroxy-4,9-estradien-3-one)(see Bocquel, J. Steroid Biochem. Molec. Biol. 45:205-215, 1993).Another GR antagonist related to RU486 is RU044 (RU43.044)17-β-hydrox-17-α-19-(4-methyl-phenyl)-androsta-4,9(11)-dien-3-one)(Bocquel, 1993, supra). See also Teutsch, Steroids 38:651-665, 1981;U.S. Pat. Nos. 4,386,085 and 4,912,097.

One embodiment includes compositions that are irreversibleanti-glucocorticoids. Such compounds include α-keto-methanesulfonatederivatives of cortisol, including cortisol-21-mesylate(4-pregnene-11-β, 17-α, 21-triol-3, 20-dione-21-methane-sulfonate anddexamethasone-21-mesylate (16-methyl-9-α-fluoro-1,4-pregnadiene-11β,17-α, 21-triol-3, 20-dione-21-methane-sulfonte). See Simons, J. SteroidBiochem. 24:25-32, 1986; Mercier, J. Steroid. Biochem. 25:11-20, 1986;U.S. Pat. No. 4,296,206.

3. Modification of the 17-Beta Side Chain Group

Steroidal anti-glucocorticoids which can be obtained by variousstructural modifications of the 17-β side chain are also used in themethods of the invention. This class includes syntheticantiglucocorticoids, such as dexamethasone-oxetanone, various 17,21-acetonide derivatives and 17-beta-carboxamide derivatives ofdexamethasone (Lefebvre, 1989, supra; Rousseau, Nature 279:158-160,1979).

4. Other Steroid Backbone Modifications

GRMs used in the various embodiments of the invention include anysteroid backbone modification which inhibits a biological responseresulting from a GR-agonist interaction. Steroid backbone antagonistscan be any natural or synthetic variation of cortisol, such as adrenalsteroids missing the C-19 methyl group, such as19-nordeoxycorticosterone and 19-norprogesterone (Wynne, Endocrinology107:1278-1280, 1980).

In general, the 11-βside chain substituent, and particularly the size ofthat substituent, can play a key role in determining the extent of asteroid's antiglucocorticoid activity. Substitutions in the A ring ofthe steroid backbone can also be important. For example,17-hydroxypropenyl side chains can, in some cases, decreaseantiglucoconicoid activity in comparison to 17-propynyl side chaincontaining compounds.

Additional glucocorticoid receptor antagonists known in the art andsuitable for practice of the invention include21-hydroxy-6,19-oxidoprogesterone (See Vicent, Mol. Pharm. 52:749-753,1997), Org31710 (See Mizutani, J Steroid Biochem Mol Biol 42(7):695-704,1992), RU43044, RU40555 (See Kim, J Steroid Biochem Mol Biol.67(3):213-22, 1998), and RU28362.

5. Nonsteroidal Anti-Glucocorticoid Receptors Modulators

Provided herein, are classes of exemplary nonsteroidal glucocorticoidreceptor modulator (GRM) and specific members of such classes that canbe used for the method disclosed herein. However, one of skill in theart will readily recognize other related or unrelated glucocorticoidreceptor modulators that can be employed in the treatment methodsdescribed herein. These include synthetic mimetics and analogs ofproteins, including partially peptidic, pseudopeptidic and non-peptidicmolecular entities. For example, oligomeric peptidomimetics useful inthe invention include (α-β-unsaturated) peptidosulfonamides,N-substituted glycine derivatives, oligo carbamates, oligo ureapeptidomimetics, hydrazinopeptides, oligosulfones and the like (See,e.g., Amour, Int. J. Pept. Protein Res. 43:297-304, 1994; de Bont,Bioorganic &Medicinal Chem. 4:667-672, 1996).

Examples of nonsteroidal GR modulators include the GR antagonistcompounds disclosed in U.S. Pat. Nos. 5,696,127; 6,570,020; and6,051,573; the GR antagonist compounds disclosed in US PatentApplication 20020077356, the glucocorticoid receptor antagonistsdisclosed in Bradley et al., J. Med. Chem. 45, 2417-2424 (2002), e.g.,4α(S)-benzyl-2(R)-chloroethynyl-1,2,3,4,4α,9,10,10α(R)-octahydro-phenanthrene-2,7-diol(“CP 394531”) and4α(S)-benzyl-2(R)-prop-1-ynyl-1,2,3,4,4α,9,10,10α(R)-octahydro-phenanthrene-2,7-diol(“CP 409069”); and the compounds disclosed in PCT InternationalApplication No, WO 96/19458, which describes non-steroidal compoundswhich are high-affinity, highly selective antagonists for steroidreceptors, such as 6-substituted-1,2-dihydro-N-protected-quinolines.

For additional compounds that can be utilized in the methods of theinvention and methods of identifying and making such compounds, see U.S.Pat. No. 4,296,206 (see above); U.S. Pat. No. 4,386,085 (see above);U.S. Pat. Nos. 4,447,424; 4,477,445; 4,519,946; 4,540,686; 4,547,493;4,634,695; 4,634,696; 4,753,932; 4,774,236; 4,808,710; 4,814,327;4,829,060; 4,861,763; 4,912,097; 4,921,638; 4,943,566; 4,954,490;4,978,657; 5,006,518; 5,043,332; 5,064,822; 5,073,548; 5,089,488;5,089,635; 5,093,507; 5,095,010; 5,095,129; 5,132,299; 5,166,146;5,166,199; 5,173,405; 5,276,023; 5,380,839; 5,348,729; 5,426,102;5,439,913; and 5,616,458; and WO 96/19458, which describes non-steroidalcompounds which are high-affinity, highly selective modulators(antagonists) for steroid receptors, such as 6-substituted-1,2-dihydroN-1 protected quinolines.

In some embodiments, the combination therapy for treating cancerinvolves a nonsteroidal GRM having a fused azadecalin backbone, aheteroaryl ketone fused azadecalin backbone, or an octahydro fusedazadecalin backbone.

Exemplary GRMs having a fused azadecalin backbone include thosedescribed in U.S. Pat. Nos. 7,928,237; and 8,461,172 and areincorporated herein in their entirety. In some cases, the GRM having afused azadecalin backbone has the following structure:

-   -   wherein    -   L¹ and L² are members independently selected from a bond and        unsubstituted alkylene;    -   R¹ is a member selected from unsubstituted alkyl, unsubstituted        heteroalkyl, unsubstituted heterocycloalkyl, —OR^(1A),        —NR^(1C)R^(1D), —C(O)NR^(1C)R^(1D), and —C(O)OR^(1A), wherein    -   R^(1A) is a member selected from hydrogen, unsubstituted alkyl        and unsubstituted heteroalkyl,    -   R^(1C) and R^(1D) are members independently selected from        unsubstituted alkyl and unsubstituted heteroalkyl,    -   wherein R^(1C) and R^(1D) are optionally joined to form an        unsubstituted ring with the nitrogen to which they are attached,        wherein said ring optionally comprises an additional ring        nitrogen;    -   R² has the formula:

-   -   wherein    -   R^(2G) is a member selected from hydrogen, halogen,        unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted        cycloalkyl, unsubstituted heterocycloalkyl, —CN, and —CF₃;    -   J is phenyl;    -   t is an integer from 0 to 5;    -   X is —S(O₂)—; and    -   R⁵ is phenyl optionally substituted with 1-5 R^(5A) groups,        wherein    -   R^(5A) is a member selected from hydrogen, halogen, —OR^(5A1),        —S(O₂)NR^(5A2)R^(5A3), —CN, and unsubstituted alkyl, wherein    -   R^(5A1) is a member selected from hydrogen and unsubstituted        alkyl, and    -   R^(5A2) and R^(5A3) are members independently selected from        hydrogen and unsubstituted alkyl,    -   or salts and isomers thereof.

Compounds containing fused azadecalin backbones can be prepared asdescribed in U.S. Pat. No. 7,928,237. For example, fused azadecalinbackbones can be prepared as described in Scheme 1, where, R⁵, R^(1A),R^(1C), R^(1D), L² and R² are as defined above in the compounds of thepresent invention. In Scheme 1, L²-R² can be replaced by a suitableprotecting group, such as BOC or benzyl, to facilitate the synthesis.Keto-ester 1 is converted directly to enone 3 by a Robinson annelationreaction involving treatment of 1 with a base (e.g. potassium or sodiumalkoxides) in an alcohol solvent (e.g. methanol, ethanol, ortest-butanol) followed by addition of methylvinyl ketone (MVK). Thereaction is typically carried out at 0-250° C.

Exemplary GRMs having a heteroaryl ketone fused azadecalin backboneinclude those described in U.S. 2014/0038926 and is incorporated hereinin its entirety. In some cases, the GRM having a heteroaryl ketone fusedazadecalin backbone has the following structure:

wherein

-   -   R¹ is a heteroaryl ring having from 5 to 6 ring members and from        1 to 4 heteroatoms each independently selected from the group        consisting of N, O and S, optionally substituted with 1-4 groups        each independently selected from R^(1a);    -   each R^(1a) is independently selected from the group consisting        of hydrogen, C₁₋₆ alkyl, halogen, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,        haloalkoxy, —CN, N-oxide, C₃₋₈ cycloalkyl, and C₃₋₈        heterocycloalkyl;    -   ring J is selected from the group consisting of a cycloalkyl        ring, a heterocycloalkyl ring, an aryl ring and a heteroaryl        ring, wherein the heterocycloalkyl and heteroaryl rings have        from 5 to 6 ring members and from 1 to 4 heteroatoms each        independently selected from the group consisting of N, O and S;    -   each R² is independently selected from the group consisting of        hydrogen, C₁₋₆ alkyl, halogen, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, C₁₋₆        haloalkoxy, C₁₋₆ alkyl-C₁₋₆ alkoxy, —CN, —OH, —NR²R^(2b),        —C(O)R^(2a), —C(O)OR^(2a), —C(O)NR^(2a)R^(2b), —SR^(2a),        —S(O)R^(2a), —S(O)₂ R^(2a), C₃₋₈ cycloalkyl, and C₃₋₈        heterocycloalkyl, wherein the heterocycloalkyl groups are        optionally substituted with 1-4 R^(2c) groups;    -   alternatively, two R² groups linked to the same carbon are        combined to form an oxo group (═O);    -   alternatively, two R² groups are combined to form a        heterocycloalkyl ring having from 5 to 6 ring members and from 1        to 3 heteroatoms each independently selected from the group        consisting of N, O and S, wherein the heterocycloalkyl ring is        optionally substituted with from 1 to 3 R^(2d) groups;

R^(2a) and R^(2b) are each independently selected from the groupconsisting of hydrogen and C₁₋₆ alkyl;

each R^(2c) is independently selected from the group consisting ofhydrogen, halogen, hydroxy, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, —CN, and—NR^(2a)R^(2b);

-   -   each R^(2d) is independently selected from the group consisting        of hydrogen and C₁₋₆ alkyl, or two R^(2d) groups attached to the        same ring atom are combined to form (═O),    -   R³ is selected from the group consisting of phenyl and pyridyl,        each optionally substituted with 1-4 R^(3a) groups;    -   each R^(3a) is independently selected from the group consisting        of hydrogen, halogen, and C₁₋₆ haloalkyl; and    -   subscript n is an integer from 0 to 3;    -   or salts and isomers thereof.

Compounds containing fused azadecalin backbones can be prepared asdescribed in Scheme 2.

Exemplary GRMs having an octahydro fused azadecalin backbone includethose described in U.S. Provisional Patent Appl. No. 61/908,333,entitled Octahydro Fused Azadecalin Glucocorticoid Receptor Modulators,filed on Nov. 25, 2013, and are incorporated herein in their entirety.In some cases, the GRM having an octahydro fused azadecalin backbone hasthe following structure:

wherein

-   -   R¹ is a heteroaryl ring having from 5 to 6 ring members and from        1 to 4 heteroatoms each independently selected from the group        consisting of N, O and S, optionally substituted with 1-4 groups        each independently selected from R^(1a);    -   each R^(1a) is independently selected from the group consisting        of hydrogen, C₁₋₆ alkyl, halogen, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,        C₁₋₆ haloalkoxy, N-oxide, and C₃₋₈ cycloalkyl;    -   ring J is selected from the group consisting of an aryl ring and        a heteroaryl ring having from 5 to 6 ring members and from 1 to        4 heteroatoms each independently selected from the group        consisting of N, O and S;    -   each R² is independently selected from the group consisting of        hydrogen, C₁₋₆ alkyl, halogen, C₁₋₆ haloalkyl, C₁₋₆ alkoxy,        haloalkoxy, C₁₋₆ alkyl-C₁₋₆ alkoxy, —CN, —OH, NR^(2a)R^(2b),        —C(O)R^(2a), —C(O)OR^(2a), —C(O)NR^(2a)R^(2b), —SR^(2a),        —S(O)R^(2a), —S(O)₂ R^(2a), C₃₋₈ cycloalkyl, and C₃₋₈        heterocycloalkyl having from 1 to 3 heteroatoms each        independently selected from the group consisting of N, O and S;    -   alternatively, two R² groups on adjacent ring atoms are combined        to form a heterocycloalkyl ring having from 5 to 6 ring members        and from 1 to 3 heteroatoms each independently selected from the        group consisting of N, O and S, wherein the heterocycloalkyl        ring is optionally substituted with from 1 to 3 R^(2c) groups;    -   R^(2a), R^(2b) and R^(2c) are each independently selected from        the group consisting of hydrogen and C₁₋₆ alkyl;    -   each R^(3a) is independently halogen; and    -   subscript n is an integer from 0 to 3;        or salts and isomers thereof.

Compounds containing octahydro fused azadecalin backbones can beprepared as described in Scheme 3.

F. Identifying Selective Glucocorticoid Receptor Modulators (SGRMS)

To determine whether a test compound is a SGRM, the compound is firstsubjected to assays to measure its ability to bind to the GR and inhibitGR-mediated activities, which determines whether the compound is aglucocorticoid receptor modulator. The compound, if confirmed to be aglucocorticoid receptor modulator, is then subjected to a specificitytest to determine whether the compound can bind specifically to GR ascompared to non GR proteins, such as the estrogen receptor, theprogesterone receptor, the androgen receptor, or the mineralocorticoidreceptor. In one embodiment, a SGRM binds to GR at a substantiallyhigher affinity, e.g., at least 10 times higher affinity, than to non-GRproteins. A SGRM may exhibit a 100 fold, 1000 fold or greaterselectivity for binding to GR relative to binding to non GR proteins.

i. Binding Assays

A test compounds' ability to bind to the glucocorticoid receptor can bemeasured using a variety of assays, for example, by screening for theability of the test compound to compete with a glucocorticoid receptorligand, such as dexamethasone, for binding to the glucocorticoidreceptor. Those of skill in the art will recognize that there are anumber of ways to perform such competitive binding assays. In someembodiments, the glucocorticoid receptor is pre-incubated with a labeledglucocorticoid receptor ligand and then contacted with a test compound.This type of competitive binding assay may also be referred to herein asa binding displacement assay. A decrease of the quantity of labeledligand bound to glucocorticoid receptor indicates that the test compoundbinds to the glucocorticoid receptor. In some cases, the labeled ligandis a fluorescently labeled compound (e.g., a fluorescently labeledsteroid or steroid analog). Alternatively, the binding of a testcompound to the glucocorticoid receptor can be measured directly with alabeled test compound. This latter type of assay is called a directbinding assay.

Both direct binding assays and competitive binding assays can be used ina variety of different formats. The formats may be similar to those usedin immunoassays and receptor binding assays. For a description ofdifferent formats for binding assays, including competitive bindingassays and direct binding assays, see Basic and Clinical Immunology 7thEdition (D. Stites and A. Terr ed.) 1991; Enzyme Immunoassay, E. T.Maggio, ed., CRC Press, Boca Raton, Fla. (1980); and “Practice andTheory of Enzyme Immunoassays,” P. Tijssen, Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers B.V.Amsterdam (1985), each of which is incorporated herein by reference.

In solid phase competitive binding assays, for example, the samplecompound can compete with a labeled analyte for specific binding siteson a binding agent bound to a solid surface. In this type of format, thelabeled analyte can be a glucocorticoid receptor ligand and the bindingagent can be glucocorticoid receptor bound to a solid phase.Alternatively, the labeled analyte can be labeled glucocorticoidreceptor and the binding agent can be a solid phase glucocorticoidreceptor ligand. The concentration of labeled analyte bound to thecapture agent is inversely proportional to the ability of a testcompound to compete in the binding assay.

Alternatively, the competitive binding assay may be conducted in theliquid phase, and any of a variety of techniques known in the art may beused to separate the bound labeled protein from the unbound labeledprotein. For example, several procedures have been developed fordistinguishing between bound ligand and excess bound ligand or betweenbound test compound and the excess unbound test compound. These includeidentification of the bound complex by sedimentation in sucrosegradients, gel electrophoresis, or gel isoelectric focusing;precipitation of the receptor-ligand complex with protamine sulfate oradsorption on hydroxylapatite; and the removal of unbound compounds orligands by adsorption on dextran-coated charcoal (DCC) or binding toimmobilized antibody. Following separation, the amount of bound ligandor test compound is determined.

Alternatively, a homogenous binding assay may be performed in which aseparation step is not needed. For example, a label on theglucocorticoid receptor may be altered by the binding of theglucocorticoid receptor to its ligand or test compound. This alterationin the labeled glucocorticoid receptor results in a decrease or increasein the signal emitted by label, so that measurement of the label at theend of the binding assay allows for detection or quantitation of theglucocorticoid receptor in the bound state. A wide variety of labels maybe used. The component may be labeled by any one of several methods.Useful radioactive labels include those incorporating ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P. Useful non-radioactive labels include those incorporatingfluorophores, chemiluminescent agents, phosphorescent agents,electrochemiluminescent agents, and the like. Fluorescent agents areespecially useful in analytical techniques that are used to detectshifts in protein structure such as fluorescence anisotropy and/orfluorescence polarization. The choice of label depends on sensitivityrequired, ease of conjugation with the compound, stability requirements,and available instrumentation. For a review of various labeling orsignal producing systems which may be used, see U.S. Pat. No. 4,391,904,which is incorporated herein by reference in its entirety for allpurposes. The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Insome cases, a test compound is contacted with a GR in the presence of afluorescently labeled ligand (e.g., a steroid or steroid analog) with aknown affinity for the GR, and the quantity of bound and free labeledligand is estimated by measuring the fluorescence polarization of thelabeled ligand.

ii. HepG2 Tyrosine Aminotransferase (TAT) Assay

Compounds that have demonstrated the desired binding affinity to GR aretested for their activity in inhibiting GR mediated activities. In oneapproach, the compounds are subject to a Tyrosine Aminotransferase Assay(TAT), which assesses the ability of a test compound to inhibit theinduction of tyrosine aminotransferase activity by dexamethasone. SeeExample 1. GR modulators that are suitable for the method disclosedherein have an IC₅₀ (half maximal inhibition concentration) of less than10 micromolar.

iii. Cell-Based Assays

Cell-based assays which involve whole cells or cell fractions containingglucocorticoid receptors can also be used to assay for a test compound'sbinding or modulation of activity of the glucocorticoid receptor.Exemplary cell types that can be used according to the methods of theinvention include, e.g., any mammalian cells including leukocytes suchas neutrophils, monocytes, macrophages, eosinophils, basophils, mastcells, and lymphocytes, such as T cells and B cells, leukemia cells,Burkitt's lymphoma cells, tumor cells (including mouse mammary tumorvirus cells), endothelial cells, fibroblasts, cardiac cells, musclecells, breast tumor cells, ovarian cancer carcinomas, cervicalcarcinomas, glioblastomas, liver cells, kidney cells, and neuronalcells, as well as fungal cells, including yeast. Cells can be primarycells or tumor cells or other types of immortal cell lines. Of course,the glucocorticoid receptor can be expressed in cells that do notexpress an endogenous version of the glucocorticoid receptor.

In some cases, fragments of the glucocorticoid receptor, as well asprotein fusions, can be used for screening. When molecules that competefor binding with the glucocorticoid receptor ligands are desired, the GRfragments used are fragments capable of binding the ligands (e.g.,dexamethasone). Alternatively, any fragment of GR can be used as atarget to identify molecules that bind the glucocorticoid receptor.Glucocorticoid receptor fragments can include any fragment of, e.g., atleast 20, 30, 40, 50 amino acids up to a protein containing all but oneamino acid of glucocorticoid receptor.

In some embodiments, a reduction in signaling triggered byglucocorticoid receptor activation is used to identify glucocorticoidreceptor modulators. Signaling activity of the glucocorticoid receptorcan be determined in many ways. For example, downstream molecular eventscan be monitored to determine signaling activity. Downstream eventsinclude those activities or manifestations that occur as a result ofstimulation of a glucocorticoid receptor. Exemplary downstream eventsuseful in the functional evaluation of transcriptional activation andantagonism in unaltered cells include upregulation of a number ofglucocorticoid response element (GRE)-dependent genes (PEPCK, tyrosineamino transferase, aromatase). In addition, specific cell typessusceptible to GR activation may be used, such as osteocalcin expressionin osteoblasts which is downregulated by glucocorticoids; primaryhepatocytes which exhibit glucocorticoid mediated upregulation of PEPCKand glucose-6-phosphate (G-6-Pase)). GRE-mediated gene expression hasalso been demonstrated in transfected cell lines using well-knownGRE-regulated sequences (e.g., the mouse mammary tumor virus promoter(MMTV) transfected upstream of a reporter gene construct). Examples ofuseful reporter gene constructs include luciferase (luc), alkalinephosphatase (ALP) and chloramphenicol acetyl transferase (CAT). Thefunctional evaluation of transcriptional repression can be carried outin cell lines such as monocytes or human skin fibroblasts. Usefulfunctional assays include those that measure IL-1 beta stimulated IL-6expression; the downregulation of collagenase, cyclooxygenase-2 andvarious chemokines (MCP-1, RANTES); LPS stimulated cytokine release,e.g., TNFα; or expression of genes regulated by NFkB or AP-1transcription factors in transfected cell-lines.

Compounds that are tested in whole-cell assays can also be tested in acytotoxicity assay. Cytotoxicity assays are used to determine the extentto which a perceived effect is due to non-glucocorticoid receptorbinding cellular effects. In an exemplary embodiment, the cytotoxicityassay includes contacting a constitutively active cell with the testcompound. Any decrease in cellular activity indicates a cytotoxiceffect.

iv. Additional Assays

Further illustrative of the many assays which can be used to identifycompositions utilized in the methods of the invention, are assays basedon glucocorticoid activities in vivo. For example, assays that assessthe ability of a putative GR modulator to inhibit uptake of 3H-thymidineinto DNA in cells which are stimulated by glucocorticoids can be used.Alternatively, the putative GR modulator can complete with3H-dexamethasone for binding to a hepatoma tissue culture GR (see, e.g.,Choi, et al., Steroids 57:313-318, 1992). As another example, theability of a putative GR modulator to block nuclear binding of3H-dexamethasone-GR complex can be used (Alexandrova et al., J. SteroidBiochem. Mol. Biol. 41; 723-725, 1992). To further identify putative GRmodulators, kinetic assays able to discriminate between glucocorticoidagonists and modulators by means of receptor-binding kinetics can alsobe used (as described in Jones, Biochem J. 204:721-729, 1982).

In another illustrative example, the assay described by Daune, Molec.Pharm, 13:948-955, 1977; and in U.S. Pat. No. 4,386,085, can be used toidentify anti-glucocorticoid activity. Briefly, the thymocytes ofadrenalectomized rats are incubated in nutritive medium containingdexamethasone with the test compound (the putative GR modulator) atvarying concentrations. ³H-uridine is added to the cell culture, whichis further incubated, and the extent of incorporation of radiolabel intopolynucleotide is measured. Glucocorticoid agonists decrease the amountof ³H-uridine incorporated. Thus, a GR modulator will oppose thiseffect.

v. Selectivity

The GR modulators selected above are then subject to a selectivity assayto determine whether they are SGRMs. Typically, selectivity assaysinclude testing a compound that binds glucocorticoid receptor in vitrofor the degree of binding to non-glucocorticoid receptor proteins.Selectivity assays may be performed in vitro or in cell based systems,as described above. Binding may be tested against any appropriatenon-glucocorticoid receptor protein, including antibodies, receptors,enzymes, and the like. In an exemplary embodiment, thenon-glucocorticoid receptor binding protein is a cell-surface receptoror nuclear receptor. In another exemplary embodiment, thenon-glucocorticoid receptor protein is a steroid receptor, such asestrogen receptor, progesterone receptor, androgen receptor, ormineralocorticoid receptor.

The selectivity of the antagonist for the GR relative to the MR can bemeasured using a variety of assays known to those of skill in the art.For example, specific antagonists can be identified by measuring theability of the antagonist to bind to the GR compared to the MR (see,e.g., U.S. Pat. Nos. 5,606,021; 5,696,127; 5,215,916; 5,071,773). Suchan analysis can be performed using either a direct binding assay or byassessing competitive binding to the purified GR or MR in the presenceof a known ligand. In an exemplary assay, cells that stably express theglucocorticoid receptor or mineralocorticoid receptor (see, e.g., U.S.Pat. No. 5,606,021) at high levels are used as a source of purifiedreceptor. The affinity of the ligand for the receptor is then directlymeasured. Those GR modulators that exhibit at least a 10 fold, 100-foldhigher affinity, often 1000-fold, for the GR relative to the MR are thenselected for use in the methods of the invention.

The selectivity assay may also include assaying the ability to inhibitGR-mediated activities, but not MR-mediated activities. One method ofidentifying such a selective GR modulator is to assess the ability of anantagonist to prevent activation of reporter constructs usingtransfection assays (see, e.g., Bocquel et al, J. Steroid Biochem Molec.Biol. 45:205-215, 1993; U.S. Pat. Nos. 5,606,021, 5,929,058). In anexemplary transfection assay, an expression plasmid encoding thereceptor and a reporter plasmid containing a reporter gene linked toreceptor-specific regulatory elements are cotransfected into suitablereceptor-negative host cells The transfected host cells are thencultured in the presence and absence of a hormone, such as cortisol oran analog thereof, able to activate the hormone responsivepromoter/enhancer element of the reporter plasmid. Next the transfectedand cultured host cells are monitored for induction (i.e., the presence)of the product of the reporter gene sequence. Finally, the expressionand/or steroid binding-capacity of the hormone receptor protein (codedfor by the receptor DNA sequence on the expression plasmid and producedin the transfected and cultured host cells), is measured by determiningthe activity of the reporter gene in the presence and absence of anantagonist. The antagonist activity of a compound may be determined incomparison to known antagonists of the GR and MR receptors (see, e.g.,U.S. Pat. No. 5,696,127). Efficacy is then reported as the percentmaximal response observed for each compound relative to a referenceantagonist compound. GR modulators that exhibits at least a 100-fold,often 1000-fold or greater, activity towards the GR relative to the MR,PR, or AR are then selected for use in the methods disclosed herein.

An exemplar SGRM that can be used in the methods disclosed herein isCORT 125134, i.e.,(R)-(1-(4-fluorophenyl)-6-((1-methyl-1H-pyrazol-4-yl)sulfonyl)-4,4a,5,6,7,8-hexahydro-1H-pyrazolo[3,4-g]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone,which has the following structure:

Another exemplar SGRM that can be used in the methods disclosed hereinis CORT125281, i.e.,((4aR,8aS)-1-(4-fluorophenyl)-6-((2-methyl-2H-1,2,3-triazol-4-yl)sulfonyl)-4,4a,5,6,7,8,8a,9-octahydro-1H-pyrazolo[3,4-a]isoquinolin-4a-yl)(4-(trifluoromethyl)pyridin-2-yl)methanone,which has the following structure:

G. Pharmaceutical Compositions and Administrationi. Formulations

In some embodiments, the present invention provides a pharmaceuticalcomposition including a pharmaceutically acceptable excipient and a SGRMand a pharmaceutically acceptable excipient and a CIC or a CIA.

Any of the SGRMs, CICs, or CIAs disclosed herein can be formulatedtogether with a pharmaceutically acceptable carrier. Such compositionsmay include one or a combination of (e.g., two or more different)antibodies, or immunoconjugates or bispecific molecules of theinvention. For example, a pharmaceutical composition of the inventioncan comprise a combination of antibodies (or immunoconjugates orbispecifics) that bind to different epitopes on the target antigen orthat have complementary activities.

The pharmaceutical compositions of the invention can be prepared andadministered in a wide variety of oral, parenteral and topical dosageforms. Oral preparations, preferably for SGRMs and CICs, includetablets, pills, powder, dragees, capsules, liquids, lozenges, gels,syrups, slurries, suspensions, etc., suitable for ingestion by thepatient. The pharmaceutical compositions can also be administered byinjection, that is, intravenously, intramuscularly, intracutaneously,subcutaneously, intraduodenally, or intraperitoneally. Also,pharmaceutical compositions can be administered by inhalation, forexample, intranasally. Additionally, nonsteroidal SGRMs can beadministered transdermally. Accordingly, the present invention alsoprovides pharmaceutical compositions including a pharmaceuticallyacceptable carrier or excipient and a SGRM or a checkpoint inhibitor.Depending on the route of administration, the active compound can becoated in a material to protect the compound from the action of enzymes,acids and other natural conditions which may inactivate the compound.

The pharmaceutical compositions of the present invention can be providedas a salt and can be formed with many acids, including but not limitedto hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,etc. Salts tend to be more soluble in aqueous or other polar solventsthan are the corresponding free base forms. In other cases, thepreparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2%sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5, that is combinedwith buffer prior to use.

For preparing pharmaceutical compositions from SGRMs or CICs, or CIAspharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. A solid carrier can beone or more substances, which may also act as diluents, flavoringagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, the activecomponent is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

The powders and tablets preferably contain from 5% or 10% to 70% of theactive compound. Suitable carriers are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of the active compound withencapsulating material as a carrier providing a capsule in which theactive component with or without other carriers, is surrounded by acarrier, which is thus in association with it. Similarly, cachets andlozenges are included. Tablets, powders, capsules, pills, cachets, andlozenges can be used as solid dosage forms suitable for oraladministration.

Suitable solid excipients are carbohydrate or protein fillers include,but are not limited to sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen. If desired,disintegrating or solubilizing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain GRmodulator mixed with a filler or binders such as lactose or starches,lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the GR modulator compounds may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycol with or without stabilizers.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution. Pharmaceutical compositions suitable forinjectable use include sterile aqueous solutions (where water soluble)or dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. In all cases, thecomposition must be sterile and must be fluid to the extent that it canbe delivered by a syringe without difficulties. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating acheckpoint inhibitor, e.g., a CIA, in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending a SGRM or a checkpointinhibitor in a vegetable oil, such as arachis oil, olive oil, sesame oilor coconut oil, or in a mineral oil such as liquid paraffin; or amixture of these. The oil suspensions can contain a thickening agent,such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents canbe added to provide a palatable oral preparation, such as glycerol,sorbitol or sucrose. These formulations can be preserved by the additionof an antioxidant such as ascorbic acid. As an example of an injectableoil vehicle, see Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. Thepharmaceutical formulations of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

The pharmaceutical compositions of the invention can be delivered bytransdermally, by a topical route, formulated as applicator sticks,solutions, suspensions, emulsions, gels, creams, ointments, pastes,jellies, paints, powders, and aerosols.

Lipid-based drug delivery systems include lipid solutions, lipidemulsions, lipid dispersions, self-emulsifying drug delivery systems(SEDDS) and self-microemuisifying drug delivery systems (SMEDDS). Inparticular, SEDDS and SMEDDS are isotropic mixtures of lipids,surfactants and co-surfactants that can disperse spontaneously inaqueous media and form fine emulsions (SEDDS) or microemulsions(SMEDDS). Lipids useful in the formulations of the present inventioninclude any natural or synthetic lipids including, but not limited to,sesame seed oil, olive oil, castor oil, peanut oil, fatty acid esters,glycerol esters, Labrafil®, Labrasol®, Cremophor®, Solutol®, Tween®,Capryol®, Capmul®, Captex®, and Peceol®.

The pharmaceutical compositions of the invention can also be deliveredas microspheres for slow release in the body. For example, microspherescan be administered via intradermal injection of drug-containingmicrospheres, which slowly release subcutaneously (see Rao, J. BiomaterSci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gelformulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, asmicrospheres for oral administration (see, e.g., Eyles, J. Pharm.Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routesafford constant delivery for weeks or months.

The pharmaceutical compositions of the invention can be provided as asalt and can be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms. In other cases, the preparationmay be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol at a pH range of 4.5 to 5.5, that is combined with bufferprior to use

In another embodiment, the pharmaceutical compositions of the inventioncan be delivered by the use of liposomes which fuse with the cellularmembrane or are endocytosed, i.e., by employing ligands attached to theliposome, or attached directly to the oligonucleotide, that bind tosurface membrane protein receptors of the cell resulting in endocytosis.By using liposomes, particularly where the liposome surface carriesligands specific for target cells, or are otherwise preferentiallydirected to a specific organ, one can focus the delivery of the GRmodulator into the target cells in vivo. (See, e.g., Al-Muhammed, J.Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol.6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989).

The pharmaceutical compositions of this disclosure may also comprise oneor more adjuvants appropriate to the indicated route of administration.If administered orally, a compound used in the methods disclosed herein,such as a CIC or SGRM, may be mixed with lactose, sucrose, starchpowder, cellulose esters of alkanoic acids, cellulose alkyl esters,talc, stearic acid, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets may contain a controlled-release formulation as maybe provided in a dispersion of active compound in hydroxypropylmethylcellulose.

ii. Dosage

Pharmaceutical compositions suitable for administuration inlcudecompositions, where the active ingredients, e.g., checkpoint inhibitorsand SGRMs are contained in an amount effective to achieve their intendedpurpose. Dosage regimens are adjusted to provide the optimum desiredresponse (e.g., a therapeutic response). For example, a single bolus maybe administered, several divided doses may be administered over time orthe dose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of factors including theactivity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the pharmacokinetics ofthe composition, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts.

The pharmaceutical compositions of the invention are preferably in unitdosage form. In such form the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, a SGRM or acheckpoint inhibitor. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of preparation,such as packeted tablets, capsules, and powders in vials or ampoules.Also, the unit dosage form can be a capsule, tablet, cachet, or lozengeitself, or it can be the appropriate number of any of these in packagedform.

The dosage regimen of the checkpoint inhibitors or the SGRMs also takesinto consideration pharmacokinetics parameters well known in the art,i.e., the rate of absorption, bioavailability, metabolism, clearance,and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochein.Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby(1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci.84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur.J. Clin. Pharmacal. 24:103-108; the latest Remington's, supra). Thestate of the art allows the clinician to determine the dosage regimenfor each individual patient, SGRM and the checkpoint inhibitor based onthe disease or condition treated.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 6000 mg, more typically 1.0 mg to 3000mg, most typically 10 mg to 300 mg. Suitable dosages also include about1 mg, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, or 2000 mg, according to the particular application and thepotency of the active component. The composition can, if desired, alsocontain other compatible therapeutic agents. Single or multipleadministrations of compositions can be administered depending on thedosage and frequency as required and tolerated by the patient.

The compositions containing a checkpoint inhibitor should provide asufficient quantity of the active component, i.e., the checkpointinhibitor, when administered alone or in combination with a SGRM, toeffectively treat the cancer, for example, in an amount being able toreduce tumor load or achieve other beneficial or desired clinicalresults related to cancer improvement. See section h, “evaluateimprovements in reducing tumor loads”. Thus, the dosage regimen may varywidely, but can be determined routinely using standard methods. In somecases, the pharmaceutical composition comprises a CIC and administrationof a daily dose of about 1 to 2,000 mg, preferably between about 10 andabout 1000 mg and most preferably between about 250 to 500 mg of theactive ingredient, may be appropriate. The daily dose can beadministered in one to four doses per day. Other dosing schedulesinclude one dose per week and one dose per two-day cycle.

In some cases, the pharmaceutical compositions contain a CIA, and thedosage (of the active component) ranges from about 0.0001 to 100 mg/kg,and more usually 0.01 to 20 mg/kg, of the host body weight. For exampledosages can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg bodyweight, 5 mg/kg body weight, 10 mg/kg body weight or within the range of0.1-20 mg/kg. An exemplary treatment regime entails administration onceper day, once per week, twice a week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months. In some cases, the treatment comprisesadministering a CIA according one of the aforementioned dosing regimensfor a first period and another of the aforementioned dosing regimens fora second period. In some cases, the treatment discontinues for a periodof time before the same or a different dosing regimen resumes. Forexample, a patient may be on a CIA dosing regimen for two weeks, off fora week, on for another two weeks, and so on. Preferred dosage regimensfor a CIA of the invention include 0.1 mg/kg body weight, 0.3 mg/kg bodyweight, 2 mg/kg body weight, 3 mg/kg body weight or 10 mg/kg viaintravenous administration, with the antibody being given using one ofthe following dosing schedules: (i) every four weeks for six dosages,then every three months; (ii) every three weeks; 3 mg/kg body weightonce followed by 1 mg/kg body weight every three weeks

In some methods, two or more CIAs with different binding specificitiesare administered simultaneously, in which case the dosage of eachantibody administered falls within the ranges indicated. CIAs areusually administered on multiple occasions. Intervals between singledosages can be, for example, weekly, monthly, every three months oryearly. Intervals can also be irregular as indicated by measuring bloodlevels of antibody to the target antigen in the patient. In somemethods, dosage is adjusted to achieve a plasma antibody concentrationof about 1-1000 μg/ml and in some methods about 25-300 μg/ml.

The compositions containing a SGRM used in the combination therapyshould provide a sufficient quantity of active agent to effectivelypotentiate the activity of the checkpoint inhibitor in treating cancer,for example, in an amount that, when combined with the therapeuticamount of a checkpoint inhibitor, can reduce tumor load or otherwisealleviate related cancer symptoms to a greater degree, or achievegreater beneficial or desired clinical results, as compared to theadministration of the checkpoint inhibitor in the same therapeuticamount without the SGRM. In some cases, the compositions provide a SGRMat an amount that renders the tumor sensitive to the checkpointinhibitor, i.e., a showing of a reduction of tumor load or other relatedclinical benefit that would not otherwise appear when the tumor istreated with the checkpoint inhibitor alone. Methods for evaluting tumorload reduction and other beneficial results are disclussed in section h“evaluate improvements in reducing tumor loads”, infra. Thus, the dosageregimen may vary widely, depending on the route of administration andtype of cancers to be treated, but can be determined routinely usingstandard methods. In some embodiments, the SGRM is administered once permonth, twice per month, three times per month, every other week, onceper week, twice per week, three times per week, four times per week,five times per week, six times per week, every other day, daily, twice aday, three times a day or more frequent.

In some cases, the daily oral dosage for the pharmaceutical compositioncontaining a SGRM can be used for the methods disclosed herein, rangesfrom about 1 to about 2000 mg per day (mg/day). In some embodiments, thedaily amount is from about 10 to 1000 mg/day, 50 to 500 mg/day, 100 to300 mg/day. Lower dosages can be used, particularly when the drug isadministered to an anatomically secluded site, such as the cerebralspinal fluid (CSF) space, in contrast to administration orally, into theblood stream, into a body cavity or into a lumen of an organ.Substantially higher dosages can be used in topical administration.Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin more detail in such publications as Remington's, supra. See alsoNieman. In “Receptor Mediated Antisteroid Action,” Agarwal, et al.,eds., De Ciruyter, N.Y. (1987). In some embodiments, the SGRM isCORT125281. In some embodiments, the SGRM is CORT 125134.

After a pharmaceutical composition including a SGRM or a checkpointinhibitor of the invention has been formulated in an acceptable carrier,it can be placed in an appropriate container and labeled for treatmentof an indicated condition. For administration of a SGRM or checkpointinhibitor, such labeling would include, e.g, instructions concerning theamount, frequency and method of administration.

iii. Combination Therapy

The method disclosed herein involves a combination therapy ofadministering both a SGRM and a checkpoint inhibitor to a subject thatsuffers from a tumor load, which, in some cases, is due to the presenceof a checkpoint inhibitor sensitive cancer. In some embodiments, thecombination therapy involves administration of a checkpoint inhibitorand a SGRM sequentially in any order during the entire or portions ofthe treatment period.

In some cases, the SGRM and the checkpoint inhibitor are administeredfollowing the same or different dosing regimen. In some cases, the SGRMis administered following a scheduled regimen while the checkpointinhibitor is administered intermittently. In some cases, the checkpointinhibitor is administered following a scheduled regimen while the SGRMis administered intermittently. In some cases, both the SGRM and thecheckpoint inhibitor are administered intermittently. In someembodiments, the SGRM is administered daily, and the checkpointinhibitor, e.g., a checkpoint inhibitor, is administered weekly orbiweekly.

In some cases, the SGRM and the checkpoint inhibitor are administeredsequentially or simultaneously once or twice per month, three times permonth, every other week, once per week, twice per week, three times perweek, four times per week, five times per week, six times per week,every other day, daily, twice a day, three times a day or more frequent,continuously over a period of time ranging from about one day to aboutone week, from about two weeks to about four weeks, from about one monthto about two months, from about two months to about four months, fromabout four months to about six months, from about six months to abouteight months, from about eight months to about 1 year, from about 1 yearto about 2 years, or from about 2 years to about 4 years, or more.

In some embodiments, the combination therapy includes co-administering aSGRM and a checkpoint inhibitor. In some embodiments, co-administrationof a checkpoint inhibitor and a SGRM involves administering the twoagents simultaneously or approximately simultaneously (e.g., withinabout 1, 5, 10,15, 20, or 30 minutes of each other).

iv. Duration

The duration of treatment with a SGRM and a checkpoint inhibitor toreduce tumor load can vary according to the severity of the condition ina subject and the subject's response to the combination therapy. In someembodiments, the SGRM and/or the checkpoint inhibitor can beadministered for a period of about 1 week to 104 weeks (2 years), moretypically about 6 weeks to 80 weeks, most typically about 9 to 60 weeks.Suitable periods of administration also include 5 to 9 weeks, 5 to 16weeks, 9 to 16 weeks, 16 to 24 weeks, 16 to 32 weeks, 24 to 32 weeks, 24to 48 weeks, 32 to 48 weeks, 32 to 52 weeks, 48 to 52 weeks, 48 to 64weeks, 52 to 64 weeks, 52 to 72. weeks, 64 to 72 weeks, 64 to 80 weeks,72 to 80 weeks, 72 to 88 weeks, 80 to 88 weeks, 80 to 96 weeks, 88 to 96weeks, and 96 to 104 weeks. Suitable periods of administration alsoinclude 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24,25, 30, 32, 35, 40, 45, 48 50, 52, 55, 60, 64, 65, 68, 70, 72, 75, 80,85, 88 90, 95, 96, 100, and 104 weeks. Generally, administration of aSGRM and/or a checkpoint inhibitor should be continued until the desiredclinically significant reduction or amelioration is observed. Treatmentwith a SGRM and a checkpoint inhibitor in accordance with the inventionmay last for as long as two years or even longer. In some embodiments,the duration of the SGRM administration is the same as that of thecheckpoint inhibitor. In some embodiments, the duration of SGRMadministration is shorter or longer than that of the checkpointinhibitor.

In some embodiments, administration of a SGRM or a checkpoint inhibitoris not continuous and can be stopped for one or more periods of time,followed by one or more periods of time where administration resumes.Suitable periods where administration stops include 5 to 9 weeks, 5 to16 weeks, 9 to 16 weeks, 16 to 24 weeks, 16 to 32 weeks, 24 to 32 weeks,24 to 48 weeks, 32 to 48 weeks, 32 to 52 weeks, 48 to 52 weeks, 48 to 64weeks, 52 to 64 weeks, 52 to 72 weeks, 64 to 72 weeks, 64 to 80 weeks,72 to 80 weeks, 72 to 88 weeks, 80 to 88 weeks, 80 to 96 weeks, 88 to 96weeks, and 96 to 100 weeks. Suitable periods where administration stopsalso include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,24, 25, 30, 32, 35, 40, 45, 48 50, 52, 55, 60, 64, 65, 68, 70, 72, 75,80, 85, 88 90, 95, 96, and 100 weeks.

H. Evaluate Improvements in Reducing Tumor Loads

The combination therapy disclosed herein can reduce tumor load. Methodsfor measuring these responses are well-known to skilled artisans in thefield of cancer therapy, e.g., as described in the Response EvaluationCriteria in Solid Tumors (“RECIST”) guidelines, available athttp://ctep.cancer.gov/protocolDevelopment/docs/recist_guideline.pdf.

In one approach, the tumor load is measured by assaying expression oftumor-specific genetic markers. This approach is especially useful formetastatic tumors or tumors that are not easily measurable, e.g., bonemarrow cancer. A tumor-specific genetic marker is a protein or othermolecule that is unique to cancer cells or is much more abundant in themas compared to non-cancer cells. For example, see WO 2006104474.Non-limiting examples of tumor-specific genetic markers include,alpha-fetoprotein (AFP) for liver cancer, beta-2-microglobulin (B2M) formultiple myeloma; beta-human chorionic gonadotropin (beta-hCG) forchoriocarcinoma and germ cell tumors; CA19-9 for pancreatic cancer, gallbladder cancer, bile duct cancer, and gastric cancer; CA-125 and HE4 forovarian cancer; carcinoembryonic antigen (CEA) for colorectal cancer;chromogranin A (CgA) for neuroendocrine tumor; fibrin/fibrinogen forbladder cancer; prostate-specific antigen (PSA) for prostate cancer; andthyroglobulin for thyroid cancer. See,http://www.cancer.gov/about-cancer/diagnosis-staging/diagnosis/tumor-markers-fact-sheet.

Methods of measuring the expression levels of a tumor-specific geneticmarker are well known. In some embodiments, mRNA of the genentic markeris isolated from the blood sample or a tumor tissue and real-timereverse transcriptase-polymerase chain reaction (RT-PCR) is performed toquantify expression of the genetic marker. In some embodiments, westernblots or immunohistochemistry analysis are performed to evaluate theprotein expression of the tumor-specific genetic marker. Typically thelevels of the tumor-specific genetic marker are measured in multiplesamples taken over time of the combination therapy of the invention, anda decrease in levels correlates with a reduction in tumor load.

In another approach, the reduction of tumor load by the combinationtherapy disclosed herein is shown by a reduction in tumor size or areduction of amount of cancer in the body. Measuring tumor size istypically achieved by imaging-based techniques. For example, computedtomography (CT) scan can provide accurate and reliable anatomicinformation about not only tumor shrinkage or growth but alsoprogression of disease by identifying either growth in existing lesionsor the development of new lesions or tumor metastasis.

In another approach, a reduction of tumor load can be assessed byfunctional and metabolic imaging techniques. These techniques canprovide earlier assessment of therapy response by observing alterationsin perfusion, oxygenation and metabolism. For example, ¹⁸F-FDG PET usesradiolabelled glucose analogue molecules to assess tissue metabolism.Tumors typically have an elevated uptake of glucose, a change in valuecorresponding to a decrease in tumor tissue metabolism indicates areduction in tumor load. Similar imaging techniques are disclosed inKang et al., Korean J. Radiol. (2012) 13(4) 371-390.

A patient receiving the combination therapy disclosed herein may exhibitvarying degrees of tumor load reduction. In some cases, a patient canexhibit a Complete Response (CR), also referred to as “no evidence ofdisease (NED)”. CR means all detectable tumor has disappeared asindicated by tests, physical exams and scans. In some cases, a patientreceiving the combination therapy disclosed herein can experience aPartial Response (PR), which roughly corresponds to at least a 50%decrease in the total tumor volume but with evidence of some residualdisease still remaining. In some cases the residual disease in a deeppartial response may actually be dead tumor or scar so that a fewpatients classified as having a PR may actually have a CR. Also manypatients who show shrinkage during treatment show further shrinkage withcontinued treatment and may achieve a CR. In some cases, a patientreceiving the combination therapy can experience a Minor Response (MR),which roughtly means a small amount of shrinkage that is more than 25%of total tumor volume but less than the 50% that would make it a PR. Insome cases, a patient receiving the combination therapy can exhibitStable Disease (SD), which means the tumors stay roughly the same size,but can include either a small amount of growth (typically less than 20or 25%) or a small amount of shrinkage (Anything less than a PR unlessminor responses are broken out. If so, then SD is defined as typicallyless 25%).

Desired beneficial or desired clinical results from the combinationtherapy may also include e.g., reduced (i.e., slowing to some extentand/or stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and/or stop) tumor metastasis; increasedresponse rates (RR); increased duration of response; relieved to someextent one or more of the symptoms associated with the cancer; decreaseddose of other medications required to treat the disease; delayedprogression of the disease; and/or prolonged survival of patients and/orimproved quality of life. Methods for evaluating these effects are wellknown and/or disclosed in, e.g., http://cancerguide.org/endpoints.htmland RECIST guidelines, supra.

All patents, patent applications, and publications discussed herein arehereby incorporated by reference in their entireties.

EXAMPLES Example 1 HEPG2 Tyrosine Aminotransferase (TAT) Assay

The following protocol describes an assay for measuring induction of TATby dexamethasone in HepG2 cells (a human liver hepatocellular carcinomacell line; ECACC, UK). HepG2 cells are cultured using MEME mediasupplemented with 10% (v/v) foetal bovine serum; 2 mM L-glutamine and 1%(v/v) NEAA at 37° C., 5%/95% (v/v) CO₂/air. The HepG2 cells are then becounted and adjusted to yield a density of 0.125×10⁶ cells/ml in RPMI1640 without phenol red, 10% (v/v) charcoal stripped FBS, 2 mML-glutamine and seeded at 25,000 cells/well in 200% μinto 96 well,sterile, tissue culture micro titre plates, and incubated at 37° C., 5%CO₂ for 24 hours.

Growth media are then removed and replaced with assay media {RPMI 1640without phenol red, 2 mM L-glutamine+10 μM forskolin}. Test compoundsare then be screened against a challenge of 100 nM dexamethasone.Compounds are then be serially half log diluted in 100% (v/v)dimethylsupfoxide from a 10 mM stock. Then an 8-point half-log dilutioncurve are generated followed by a 1:100 dilution into assay media togive a 10× final assay of the compound concentration, this results infinal assay of the compound concentration that ranged 10 to 0.003 μM in0.1% (v/v) dimethylsulfoxide.

Test compounds are pre-incubated with cells in micro-titre plates for 30minutes at 37° C., 5/95 (v/v) CO₂/air, before the addition of 100 nMdexamethasone and then subsequently for 20 hours to allow optimal TATinduction.

HepG2 cells are then lysed with 30 μl of cell lysis buffer containing aprotease inhibitor cocktail for 15 minutes at 4° C., 155 μl of substratemixture can then be added containing 5.4 mM Tyrosine sodium salt, 10.8mM alpha ketoglutarate and 0.06 mM pyridoxal 5′ phosphate in 0.1Mpotassium phosphate buffer (pH 7.4). After 2 hours incubation at 37° C.the reaction can be terminated by the addition of 15 μl of 10M aqueouspotassium hydroxide solution, and the plates incubated for a further 30minutes at 37° C. The TAT activity product can be measured by absorbanceat λ 340 nm.

IC₅₀ values can be calculated by plotting % inhibition (normalised to100 nM dexamethasone TAT stimulation) v. compound concentration andfitting the data to a 4 parameter logistic equation. IC₅₀ values canconverted to Ki (equilibrium dissociation constant) using the Cheng andPrusoff equation, assuming the antagonists were competitive inhibitorswith respect to dexamethasone.

Example 2 Quantify the GR Expression in Cancer Types

Formalin-fixed paraffin-embedded (FFPE) tumor tissue sections of 4-5 μmthickness are cut onto positively charged slides (Fisher ProbeOn Plus™,Thermo Fisher Scientific), baked at 65° C. (dry heat) for 1 hour lessthan 1 week before use, deparaffinized in four changes of 100% xylene,and rehydrated with a graded ethanol series (100%, 70%, 30%) todistilled water.

Prepared slides are incubated for 20 minutes at 0.98° C. in Citra PlusTarget Retrieval Solution (BioGenex [Cat #: HK #080-9K], Fremont,Calif., USA), using a commercial steamer as the heat source (Black andDecker HS1000 model steamer; Black and Decker, Baltimore, Md., USA).After cooling for 5 minutes, automated staining is performed using aTechMate™ 500 or 1000 automated IMC staining platform (RocheDiagnostics, Oro Valley, Ariz., USA) and WorkMate™ software, version3.96. This automated platform uses a capillary gap process29 for allreagent changes, including antibody incubation, detection steps up toand including counterstaining, and intervening washes. All proceduresare carried out at room temperature (25° C.). Following a 15-minuteincubation with a protein serum block (QualTek Proprietary), slides areincubated with the anti-GR antibody, clone D8H2 (Cell SignalingTechnology [#3660S]) at a concentration of 1:1,750 in a primary antibodydiluent (QualTek Proprietary) for 1 hour.

The Rabbit Polink2+ HRP (horseradish peroxidase) reagents kit (GoldenBridge International [GBI], Cat #: D39-110, Los Angeles, Calif., USA),which is biotin-independent and reduces the potential for background ornonspecific staining from endogenous biotin, is used for primaryantibody detection. The steps included are a 25-minute incubation withRabbit Polink2+ secondary, a 7.5-minute peroxidase blocking step (3% USPH2O2, with ˜0.02% v/v Tween-20 added), a 25-minute incubation withRabbit Polink2+ HRP conjugated polymer, and a 15-minute incubation withGBI (Cat #: C09-100) 3,3′-diaminobenzidine (DAB) chromogen. Between allincubation steps, slides are extensively washed with tris-bufferedsaline containing 0.02% v/v Tween®-20 detergent (TBST) (Thermo FisherScientific). The slides are counterstained with hematoxylin for 1minute, rinsed in distilled water, dehydrated off platform in an ethanolseries (95%, 100%) and four changes of 100% xylene, and permanentlysealed with coverslips (Cytoseal™ XYL mounting media, Thermo FisherScientific).

A percent score is used to semiquantitatively assess tumor GR expressionin samples with at least 100 viable invasive carcinoma cells. Theintensity of nuclei staining is reported based on the H-score methodusing 0 for negative staining, 1+ for weak staining, 2+ for moderatestaining, and 3+ for strong staining. The H-score is calculated bymultiplying the staining intensity by the proportion of cells with thatintensity. For example, a sample with 20% of cells having weak stainingand 2% of cells having moderate staining would have an H-score of 24[(20×1)+(2×2)=24]. For this assay, GR positivity is defined as 10%nuclear staining of tumor cells at any intensity A board-certifiedpathologist scores nuclear tumor staining in the total area of viabletissue section available; areas of cytoplasmic or stromal staining, insitu carcinoma, necrosis, or obviously poorly fixed areas of tissue arenot evaluated.

Example 3 Reducing Tumor Growth Using the Combination Therapy of CORT125134 AND ANTI PD-1

Suspensions of mouse MC38 cancer cells were injected subcutaneously intothe left flank of 5-6 week old immunocompetent female mice (C57BL/6), 1million cells per mouse. Tumors were allowed to grow until they reach avolume of 100 mm³. Mice were then grouped into three groups, ten (10)per group. Group I was dosed with the anti-PD-1 vehicle (PBS, 10 ml/kg)i. p. twice a week and the CORT125134 vehicle p.o. (10% DMSO, 0.1% Tween80 and 89.9% HPMC (0.5%), 10 ml/kg) daily. Group II was dosed with themouse anti PD-1 antibody (clone RPM1-14, 10 mg/kg) i. p, twice a week.Group III was dosed with the mouse anti PD-1 antibody (clone RPM1-14, 10mg/kg) i. p. twice a week and CORT125134 (a SGRM) (30 mg/kg) orally on adaily basis.

The longest (L) and shortest (S) diameters of the tumors were measuredthree times a week with electronic calipers and tumor volume wascalculated using the formula for an ellipsoid sphere: S²×L×(0.5). Thetumor growth data are shown in FIG. 1, in which the mean tumor volume ascompared to the mean pre-dosing tumor volume for each group of mice isplotted against the number of days of tumor growth since initiation ofthe treatment. The result shows that the combination of anti PD-1 andCORT125134 is superior to both the anti PD-1 group and the vehicle groupin reducing tumor growth.

Example 4 Reducing Tumor Growth Using the Combination Therapy ofCORT125281 and Anti PD-1

Suspensions of mouse A20 cancer cells were injected subcutaneously intothe left flank of 5-6 week old immunocompetent female mice (C57BL/6), ½million cells per mouse. Tumors were allowed to grow until they reacheda volume of 100 mm³. Mice were then grouped into three groups, ten (10)per group. Group I was dosed with the anti-PD-1 vehicle (PBS, 10 ml/kg)i. p. twice a week and the CORT125281 vehicle p.o. (10% DMSO, 0.1% Tween80 and 89.9% HPMC (0.5%), 10 ml/kg) daily. Group II was dosed with themouse anti PD-1 antibody (clone RPM1-14, 10 mg/kg) i. p. twice a week.Group III was dosed with the mouse anti PD-1 antibody (clone RPM1-14, 10mg/kg) i. p. twice a week and CORT125281 (a SGRM) (30 mg/kg) orally on adaily basis.

The longest (L) and shortest (S) diameters of the tumors were measuredthree times a week with electronic calipers and tumor volume wascalculated using the formula for an ellipsoid sphere: S²×L×(0.5). Thetumor growth data are shown in FIG. 2, in which the mean tumor volumefor each group of mice is plotted against the number of days of tumorgrowth since initiation of the treatment. The result shows that thecombination of anti PD-1 and CORT125281 is superior to both the antiPD-1 group and the vehicle group in reducing tumor growth.

Example 5 Reducing Tumor Growth Using the Combination Therapy ofCORT125134 or CORT125281 and Anti CTLA4

Suspensions of mouse CT26 cancer cells were injected subcutaneously intothe left flank of 6-7 week old immunocompetent female mice (C57BL/6), ½million cells per mouse. Tumors were allowed to grow until they reacheda volume of 50-100 mm³. Mice were then grouped into three groups, ten(10) per group. Group I was dosed with the anti-CTLA4 vehicle (PBS, 10ml/kg) i. p. twice a week and the CORT125134 vehicle p.o. (10% DMSO,0.1% Tween 80 and 89.9% HPMC (0.5%), 10 ml/kg) daily. Group II was dosedwith the mouse anti CTLA4 antibody (clone 9D9, 10 mg/kg) i. p. twice aweek. Group III was dosed with the mouse anti CTLA4 antibody (clone 9D9,10 mg/kg) i. p. twice a week and CORT125134 (a SGRM) (30 mg/kg) orallyon a daily basis. Group IV was dosed with the mouse anti CTLA4 antibody(clone 9D9, 10 mg/kg) i. p. twice a week and CORT125281 (a SGRM) (30mg/kg) orally on a daily basis.

The longest (L) and shortest (S) diameters of the tumors were measuredthree times a week with electronic calipers and tumor volume wascalculated using the formula for an ellipsoid sphere: S²×L×(0.5). Thetumor growth data are shown in FIG. 3, in which the mean tumor volumefor each group is plotted against the number of days of tumor growthsince initiation of the treatment. The result shows that the combinationof anti CTLA4 with CORT125134 and the combination of anti CTLA4 withCORT125281 are superior to both the anti CTLA4 group and the vehiclegroup in reducing tumor growth.

Example 6 Treating a Breast Cancer Patient with the Combination Therapy

A patient suffering from breast cancer is treated with prembrolizumab ata dose of 3 mg/kg every 2 weeks and CORT125134 at a dose of 200 mg oncea day for eight weeks. Her tumor load is monitored using enhancedmagnetic resonance imaging before, during and after the treatment. Theimaging result indicate that the size of the tumor has graduallydecreased, and the reduction is more than 20% at the end of thetreatment period.

What is claimed is:
 1. A method for reducing tumor load in a patient hosting a tumor, the method effective to potentiate the activity of the antibody checkpoint inhibitor effective to provide superior tumor load reduction as compared to the tumor load reduction that would be provided by said antibody checkpoint inhibitor alone, comprising: administering to said patient hosting a tumor a therapeutic amount of the antibody checkpoint inhibitor and between about 10 milligrams (mg) to about 1000 mg per day of a selective glucocorticoid receptor antagonist (SGRA) effective to provide tumor load reduction at least 10% greater than the tumor load reduction provided by the antibody checkpoint inhibitor alone, wherein the antibody checkpoint inhibitor is selected from lambrolizumab, nivolumab, AMP-224, pidilizumab, ipilimumab, tremelimumab, MEDI4736, MPDL3280A and BMS-936559 (MDX-1105), wherein said SGRA is a compound that has a heteroaryl-ketone fused azadecalin backbone or an octahydro fused azadecalin backbone, and that binds the glucocorticoid receptor (GR) with a GR binding affinity that is at least ten-fold greater than its binding affinity for the mineralocorticoid receptor, said GR binding being able to inhibit GR-mediated activity, and where the SGRA is administered in an amount effective to potentiate the activity of the antibody checkpoint inhibitor effective to provide at least 10% greater tumor load reduction in the patient as compared to the tumor load reduction provided by the antibody checkpoint inhibitor alone.
 2. The method of claim 1, wherein the tumor load is due to the presence of a checkpoint inhibitor-sensitive tumor.
 3. The method of claim 1 wherein the checkpoint inhibitor is an antibody effective against PD-1 selected from lambrolizumab, nivolumab, AMP-224, and pidilizumab.
 4. The method of claim 1 wherein the checkpoint inhibitor is an antibody effective against CTLA-4 selected from ipilimumab and tremelimumab.
 5. The method of claim 1 wherein the checkpoint inhibitor is an antibody effective against PD-L1 or PD-L2 selected from MEDI4736, MPDL3280A and BMS-936559 (MDX-1105).
 6. The method of claim 1 wherein the SGRA is CORT125134


7. The method of claim 1, wherein the SGRA is CORT125281


8. The method of claim 1 wherein the cancer expresses the glucocorticoid receptor (GR⁺).
 9. The method of claim 1 wherein the cancer is a GR⁺ cancer and wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, Melanoma, Sarcoma, Renal cell cancer, Head and Neck cancer, Hepatocellular cancer, Glioblastoma, Cervical cancer, neuroendocrine cancer, Bladder cancer, Prostate cancer, Esophageal cancer, mesothelioma, Lung cancer, Ovarian cancer, Pancreas cancer, Gall bladder cancer, Gastric cancer, Endometrial cancer, and colon cancer.
 10. The method of claim 7 wherein the SGRA is CORT125281 and the checkpoint inhibitor is an antibody effective against PD-1 selected from the group of anti-PD-1 antibody checkpoint inhibitors consisting of lambrolizumab, nivolumab, AMP-224, and pidilizumab.
 11. The method of claim 6 wherein the SGRA is CORT125134 and the checkpoint inhibitor is an antibody effective against PD-1 selected from the group of anti-PD-1 antibody checkpoint inhibitors consisting of lambrolizumab, nivolumab, AMP-224, and pidilizumab.
 12. The method of claim 1 wherein the SGRA is CORT125281 or CORT125134 and the checkpoint inhibitor is an antibody effective against CTLA-4 selected from the group of anti-CTLA-4 antibody checkpoint inhibitors consisting of ipilimumab and tremelimumab.
 13. The method of claim 1 wherein the SGRA is CORT125281 or CORT125134 and the checkpoint inhibitor is an antibody effective against PDL-1 or PDL-2 selected from the group of anti-PD-L1 or anti-PD-L2 antibody checkpoint inhibitors consisting of MEDI4736, MPDL3280A and BMS-936559 (MDX-1105). 